Pharmaceutical composition for treating and/or preventing cancer

ABSTRACT

According to the present invention, a cancer antigen protein to be specifically expressed on the surfaces of cancer cells is identified and thus the use of an antibody targeting the cancer antigen protein as an agent for treating and/or preventing a cancer is provided. Specifically, the present invention provides a pharmaceutical composition for treating and/or preventing a cancer, which comprises an antibody comprising a heavy chain variable region that comprises SEQ ID NOS: 39, 40, and 41 and a light chain variable region that comprises SEQ ID NOS: 43, 44, and 45 or a fragment thereof as an active ingredient and having immunological reactivity with a CAPRIN-1 protein.

TECHNICAL FIELD

The present invention relates to a novel pharmaceutical use of anantibody against CAPRIN-1 or a fragment thereof, as an agent, fortreating and/or preventing a cancer.

BACKGROUND ART

Cancer is the leading cause of death. Currently conducted therapycomprises mainly surgical therapy in combination with radiation therapyand chemotherapy. In spite of the development of new operativeprocedures and the discovery of new anticancer agents in recent years,cancer treatment results have not been much improved recently, excludingthat for some types of cancer. Recent advances in molecular biology orcancer immunology lead to identification of antibodies specificallyreacting with cancer, cancer antigens to be recognized by cytotoxic Tcells, genes encoding cancer antigens, and the like. Demands on specificcancer therapies targeting cancer antigens are increasing (Non-patentLiterature 1).

In cancer therapy, it is desirable that peptides, polypeptides, orproteins recognized as antigens be almost absent in normal cells, butthey be present specifically in cancer cells, in order to alleviate sideeffects. In 1991, Boon et al., (Ludwig Institute for Cancer Research,Belgium) isolated a human melanoma antigen MAGE1 recognized byCD8-positive T cells by the cDNA expression cloning method usingautologous cancer cell lines and cancer-reactive T cells (Non-patentLiterature 2). Thereafter, the SEREX (serological identification ofantigens by recombinant expression cloning) method that comprisesidentifying tumor antigens recognized by antibodies that are produced invivo in response to autologous cancer of a cancer patient by geneexpression cloning techniques was reported (Non-patent Literature 3 andPatent Literature 1). With the use of this method, some cancer antigens,which are almost never expressed in normal cells but are specificallyexpressed in cancer cells, were isolated (Non-patent Literatures 4-9).Furthermore, clinical trials were conducted with cell therapiestargeting some cancer antigens using immunocytes specifically reactivewith cancer antigens, or cancer-specific immunotherapies using vaccinesor the like containing cancer antigens.

Meanwhile, in recent years, various antibody medicines which targetantigenic proteins on cancer cells for cancer treatment have appearedthroughout the world. Antibody medicines exhibit some pharmacologicaleffects as cancer specific therapeutic agents and are thus attractingattention. However, most antigen proteins to be targeted are alsoexpressed in normal cells, so that not only cancer cells, but alsonormal cells expressing antigens are also damaged as a result ofantibody administration. The resulting side effects cause for concern.Therefore, it is expected that identification of cancer antigens thatare specifically expressed on the surface of a cancer cell and use ofantibodies targeting the cancer antigens as pharmaceuticals will realizetreatment with antibody medicines with lower side effects.

Cytoplasmic- and proliferation-associated protein 1 (CAPRIN-1) isexpressed when normal cells at the resting phase are activated orundergo cell division, and it is an intracellular protein known to formintracellular stress granules with RNA within cells, so as to beinvolved in mRNA transport and translational regulation. Meanwhile, manyother names that represent CAPRIN-1 exist, such as GPI-anchored membraneprotein 1 or membrane component surface marker 1 protein (M11S1), as ifsuch proteins had been known to be cell membrane proteins. These namesoriginated from a report that the gene sequence of CAPRIN-1 is amembrane protein having a GPI-binding region and expressed in colorectalcancer cells (Non-patent Literature 10). However, the gene sequence ofCAPRIN-1 provided in this report was later revealed to be wrong. Thefollowing has recently been reported; i.e., deletion of a singlenucleotide in the gene sequence of CAPRIN-1 registered at GenBank or thelike causes a frame shift, so that 80 amino acids are lost from theC-terminus, resulting in generation of an artifact (74 amino acids)which corresponds to the GPI-binding portion in the previous report, andadditionally, another error is also present 5′ of the gene sequence, sothat 53 amino acids were lost from the N-terminus (Non-patent Literature11). It has been also recently reported that the protein encoded by thegene sequence of CAPRIN-1 registered at GenBank or the like is not acell membrane protein (Non-patent Literature 11).

In addition, on the basis of the report of Non-patent Literature 10 thatCAPRIN-1 is a cell membrane protein, Patent Literatures 2 and 3 describethat CAPRIN-1 (as a cell membrane protein) under the name of M11S1 canbe used as a target of an antibody medicine in cancer therapy, althoughworking examples do not describe treatment using an antibody against theprotein. However, as reported in Non-patent Literature 11, it has beencommonly believed from the time of the filing of Patent Literature 2 todate that CAPRIN-1 is not expressed on the surface of a cell. Thecontents of Patent Literatures 2 and 3 based only on incorrectinformation that CAPRIN-1 is a cell membrane protein should not clearlybe understood as common general knowledge for persons skilled in theart.

PRIOR ART LITERATURE Patent Literature

-   Patent Literature 1: U.S. Pat. No. 5,698,396-   Patent Literature 2: US2008/0075722-   Patent Literature 3: WO2005/100998

Non-Patent Literature

-   Non-patent Literature 1: Tsuyoshi Akiyoshi, “Gan To Kagaku-Ryoho    (Cancer and Chemotherapy),” 1997, Vol. 24, p 551-519 (Cancer and    Chemotherapy Publishers, Inc., Japan)-   Non-patent Literature 2: Bruggen P. et al., Science, 254: 1643-1647    (1991)-   Non-patent Literature 3: Proc. Natl. Acad. Sci. U.S.A, 92:    11810-11813 (1995)-   Non-patent Literature 4: Int. J. Cancer, 72: 965-971 (1997)-   Non-patent Literature 5: Cancer Res., 58: 1034-1041 (1998)-   Non-patent Literature 6: Int. J. Cancer, 29: 652-658 (1998)-   Non-patent Literature 7: Int. J. Oncol., 14: 703-708 (1999)-   Non-patent Literature 8: Cancer Res., 56: 4766-4772 (1996)-   Non-patent Literature 9: Hum. Mol. Genet 6: 33-39, 1997-   Non-patent Literature 10: J. Biol. Chem., 270: 20717-20723, 1995-   Non-patent Literature 11: J. Immunol., 172: 2389-2400, 2004

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

Objects of the present invention are to identify a cancer antigenprotein specifically expressed on the surface of a cancer cell and toprovide the use of an antibody targeting the cancer antigen protein asan agent for treating and/or preventing a cancer.

Means for Solving the Problem

As a result of intensive studies, the present inventors have nowobtained a cDNA encoding a protein that binds to an antibody existing insera from dogs with breast cancer by the SEREX method using both cDNAlibraries prepared from dog testis tissues and sera of dogs with breastcancer. The present inventors have now further prepared CAPRIN-1proteins having the even-numbered amino acid sequences of SEQ ID NOS: 2to 30 and antibodies against such CAPRIN-1 proteins based on theobtained dog gene and the corresponding human, cattle, horse, mouse, andchicken homologous genes. Thus, the present inventors have now foundthat CAPRIN-1 is specifically expressed in breast cancer, brain tumor,leukemia, lymphoma, lung cancer, uterine cervix cancer, bladder cancer,esophageal cancer, colorectal cancer, gastric cancer, and renal cancercells, and that a portion of the CAPRIN-1 protein is specificallyexpressed on the surface of each cancer cell. The present inventors havethus now found that an antibody or antibodies against the portion ofCAPRIN-1 expressed on the surface of each cancer cell is/are cytotoxicto the CAPRIN-1-expressing cancer cells. On the basis of these findings,the present invention as described below was completed.

The present invention has the following characteristics.

The present invention provides a pharmaceutical composition for treatingand/or preventing a cancer, comprising an antibody that comprises aheavy chain variable region comprising SEQ ID NO: 39, 40, and 41 and alight chain variable region comprising SEQ ID NO: 43, 44, and 45 or afragment thereof as an active ingredient and having immunologicalreactivity with a CAPRIN-1 protein.

In an embodiment, the above cancer is breast cancer, brain tumor,leukemia, lymphoma, lung cancer, uterine cervix cancer, bladder cancer,esophageal cancer, colorectal cancer, gastric cancer, or renal cancer.

In another embodiment, the above antibody is a human antibody, humanizedantibody, chimeric antibody, single chain antibody, or bispecificantibody.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2010-023452, which is a priority literature of the present application.

Effects of the Invention

The antibody against CAPRIN-1 used in the present invention is cytotoxicto cancer cells. As such, the antibody against CAPRIN-1 is useful fortreating or preventing cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression patterns of genes encoding CAPRIN-1 proteinsin normal tissues and tumor cell lines. Reference No. 1 indicates theexpression patterns of genes encoding CAPRIN-1 proteins, and ReferenceNo. 2 indicates the expression patterns of GAPDH genes.

FIG. 2 shows the cytotoxicity of an anti-CAPRIN-1 monoclonal antibody #1(that is reactive with the surfaces of the cancer cells) against theMDA-MB-157 breast cancer cell line expressing CAPRIN-1. Reference No. 3indicates the activity exhibited when the anti-CAPRIN-1 monoclonalantibody #1 was added. Reference No. 4 indicates the activity exhibitedwhen PBS was added instead of the antibodies.

FIG. 3 shows the anti-tumor effects of the anti-CAPRIN-1 monoclonalantibody #1 (that is reactive with the surfaces of cancer cells) againstBalb/c mice into which the 4T1 mouse breast cancer cell line expressingCAPRIN-1 was transplanted. Reference No. 5 indicates the tumor size of amouse to which the anti-CAPRIN-1 monoclonal antibody #1 wasadministered. Reference No. 6 indicates the tumor size of a mouse towhich PBS was administered instead of the antibodies.

MODE FOR CARRYING OUT THE INVENTION

The anti-tumor activity of an antibody against a polypeptide representedby any of the even-numbered sequences of SEQ ID NOS: 2 to 30 used in thepresent invention can be evaluated by examining in vivo suppression oftumor growth in animals with cancer, or, examining whether or not theantibody exhibits cytotoxicity via immunocytes or complements to tumorcells expressing the polypeptide in vitro, as described later.

In the context, the nucleotide sequences of polynucleotides encodingproteins comprising the even-numbered amino acid sequences (i.e., SEQ IDNOS: 2, 4, 6 . . . 28, 30) of SEQ ID NOS: 2 to 30 are represented by theodd-numbered sequences (i.e., SEQ ID NOS: 1, 3, 5 . . . 27, 29) of SEQID NOS: 1 to 29.

The amino acid sequences that are represented by SEQ ID NOS: 6, 8, 10,12, and 14 in the Sequence Listing disclosed herein are the amino acidsequences of CAPRIN-1 isolated as polypeptides, which bind to antibodiesspecifically existing in serum from a dog with cancer, through the SEREXmethod using a cDNA library from dog testis tissue and the serum of adog with breast cancer. The amino acid sequences represented by SEQ IDNOS: 2 and 4 are the amino acid sequences of CAPRIN-1 isolated as humanhomologues. The amino acid sequence represented by SEQ ID NO: 16 is theamino acid sequence of CAPRIN-1 isolated as a cattle homologue. Theamino acid sequence represented by SEQ ID NO: 18 is the amino acidsequence of CAPRIN-1 isolated as a horse homologue. The amino acidsequences represented by SEQ ID NOS: 20 to 28 are the amino acidsequences of CAPRIN-1 isolated as mouse homologues. The amino acidsequence represented by SEQ ID NO: 30 is the amino acid sequence ofCAPRIN-1 isolated as a chicken homologue (see Example 1 describedlater). CAPRIN-1 is known to be expressed when normal cells in theresting phase are activated or give rise to cell division.

It was known that CAPRIN-1 was not expressed on cell surfaces. However,as a result of the examination by the present inventors, it has now beenrevealed that a portion of the CAPRIN-1 protein is expressed on thesurfaces of various cancer cells. It has thus been revealed that anantibody having immunological reactivity with a partial region of aCAPRIN-1 protein that is expressed on cancer cell surfaces orspecifically recognizing the region (that is, specifically binding tothe region) and comprising a heavy chain variable region that comprisesSEQ ID NO: 39, 40, and 41 and a light chain variable region thatcomprises SEQ ID NO: 43, 44, and 45 exhibits anti-tumor activity.

The above-described anti-CAPRIN-1 antibody used in the present inventionmay be any type of monoclonal antibody as long as it can exhibitanti-tumor activity. Examples of such an antibody include recombinantantibodies, such as a synthetic antibody, a multispecific antibody, ahumanized antibody, a chimeric antibody, and a single chain antibody(scFv), a human antibody, and antibody fragments thereof, such as Fab,F(ab′)₂, and Fv. These antibodies and fragments thereof can be preparedby methods known by persons skilled in the art. Also, when a subject isa human, human antibodies or humanized antibodies are desired in orderto avoid or suppress rejection.

The term “specifically binding to a partial region of a CAPRIN-1protein” as used herein means that “the antibody specifically binds to aspecific region of a CAPRIN-1 protein without substantially binding toprotein portions other than such a region.”

The anti-tumor activity of an antibody that can be used in the presentinvention can be evaluated as described below by examining in vivo thesuppression of the tumor growth in animals with cancer, or, by examiningwhether or not it exhibits in vitro an activity of cytotoxicity, whichis mediated by immunocytes or complements, to tumor cells expressing thepolypeptide.

Furthermore, examples of the subject for cancer treatment and/orprevention in the present invention include mammals, such as humans, petanimals, domestic animals, and animals for competition. A preferablesubject is a human.

Preparation of antigens and antibodies and pharmaceutical compositionsrelating to the present invention are described below.

<Preparation of Antigens for Antibody Preparation>

Proteins or fragments thereof to be used as sensitizing antigens forobtaining anti-CAPRIN-1 antibodies used in the present invention may bederived from any animal species without particular limitation, such ashumans, dogs, cattle, horses, mice, rats, and chickens. However,proteins or fragments thereof are preferably selected in considerationof compatibility with parent cells used for cell fusion. In general,mammal-derived proteins are preferred and, in particular, human-derivedprotein is preferred. For example, when CAPRIN-1 is human CAPRIN-1, thehuman CAPRIN-1 protein, a partial peptide thereof, or cells expressinghuman CAPRIN-1 can be used.

The nucleotide sequences and the amino acid sequences of human CAPRIN-1and homologues thereof can be obtained by accessing GenBank (NCBI,U.S.A.) and using an algorithm such as BLAST or FASTA (Karlin andAltschul, Proc. Natl. Acad. Sci. U.S.A., 90: 5873-5877, 1993; Altschulet al., Nucleic Acids Res. 25: 3389-3402, 1997).

In the present invention, on the basis of the nucleotide sequence (SEQID NO: 1 or 3) or the amino acid sequence (SEQ ID NO: 2 or 4) of humanCAPRIN-1, a target nucleic acid or a target protein comprises a sequencehaving 70% to 100%, preferably 80% to 100%, more preferably 90% to 100%,even more preferably 95% to 100% (e.g., 97% to 100%, 98% to 100%, 99% to100%, or 99.5% to 100%) sequence identity with the nucleotide sequenceor the amino acid sequence of the ORF or the mature portion of humanCAPRIN-1. As use herein, the term “% sequence identity” refers to apercentage (%) of identical amino acids (or nucleotides) relative to thetotal number of amino acids (or nucleotides), when two sequences arealigned to achieve the highest similarity with or without introductionof gaps.

The length of a fragment of CAPRIN-1 protein ranges from the amino acidlength of an epitope (antigenic determinant), which is the minimum unitrecognized by an antibody, to a length less than the full length of theprotein. The term “epitope” refers to a polypeptide fragment havingantigenicity or immunogenicity in mammals, preferably in humans, and theminimum unit of the epitope consists of about 7 to 12 amino acids (e.g.,8 to 11 amino acids).

The polypeptides comprising the above-mentioned human CAPRIN-1 proteinor partial peptides of the protein, can be synthesized by a chemicalsynthesis method, such as the Fmoc method (fluorenylmethyloxycarbonylmethod) or the tBoc method (t-butyloxycarbonyl method) (Edited by TheJapanese Biochemical Society, Seikagaku Jikken Koza (BiochemicalExperimental Lecture Series) 1, Protein Chemistry IV, ChemicalModification and Peptide Synthesis, TOKYO KAGAKU DOZIN (Japan), 1981).Alternatively, the above-mentioned polypeptides may also be synthesizedby conventional methods using various commercially available peptidesynthesizers. Furthermore, with the use of known genetic engineeringtechniques (e.g., Sambrook et al., Molecular Cloning, 2^(nd) Edition,Current Protocols in Molecular Biology (1989), Cold Spring HarborLaboratory Press, Ausubel et al., Short Protocols in Molecular Biology,3^(rd) Edition, A compendium of Methods from Current Protocols inMolecular Biology (1995), John Wiley & Sons), a polynucleotide encodingthe above polypeptide is prepared and then incorporated into anexpression vector, which is subsequently introduced into a host cell inorder to produce a polypeptide of interest in the host cell, and thenrecover it.

The polynucleotides encoding the above polypeptides can be easilyprepared by known genetic engineering techniques or conventionaltechniques using a commercially available nucleic acid synthesizer. Forexample, DNA comprising the nucleotide sequence of SEQ ID NO: 1 can beprepared by PCR using a human chromosomal DNA or cDNA library, as atemplate, and a pair of primers designed to be able to amplify thenucleotide sequence represented by SEQ ID NO: 1. PCR conditions can beappropriately determined. For example, PCR conditions compriseconducting 30 cycles of the reaction cycle of: denaturation at 94° C.for 30 seconds; annealing at 55° C. for 30 seconds to 1 minute; andextension at 72° C. for 2 minutes, using a thermostable DNA polymerase(e.g., Taq polymerase or Pfu polymerase) and PCR buffer containing Mg²⁺,followed by reacting at 72° C. for 7 minutes. However, the PCRconditions are not limited to the above example. PCR techniques,conditions, and the like are described in Ausubel et al., ShortProtocols in Molecular Biology, 3^(rd) Edition, A compendium of Methodsfrom Current Protocols in Molecular Biology (1995), John Wiley & Sons(particularly Chapter 15).

Also, on the basis of the nucleotide sequence and amino acid sequenceinformation represented by SEQ ID NOS: 1 to 30 in the Sequence Listingdescribed herein, appropriate probes or primers are prepared, and then acDNA library of a human or the like is screened using them, so thatdesired DNA can be isolated. A cDNA library is preferably constructedfrom cells, organs or tissues, which express proteins havingeven-numbered sequences of SEQ ID NOS: 2 to 30. Examples of such cellsor tissues include cells or tissues derived from testis, and cancers ortumors, such as leukemia, breast cancer, lymphoma, brain tumor, lungcancer, colorectal cancer, and the like. Procedures such as thepreparation of probes or primers, construction of a cDNA library,screening of a cDNA library, and cloning of target genes are known by aperson skilled in the art and can be carried out by the methodsdescribed in Sambrook et al., Molecular Cloning, 2^(nd) Edition, CurrentProtocols in Molecular Biology (1989), Ausbel et al., (above), and thelike. DNA encoding a human CAPRIN-1 protein or a partial peptide thereofcan be obtained from the thus obtained DNA.

The host cells may be any cells, as long as they can express theabove-mentioned polypeptide. Examples of prokaryotic cells include, butare not limited to, Escherichia coli and the like. Examples ofeukaryotic cells include, but are not limited to, mammalian cells, suchas monkey kidney cells (COS1) and Chinese hamster ovary cells (CHO),human fetal kidney cell line (HEK293), fetal mouse skin cell line(NIH3T3), yeast cells such as budding yeast and fission yeast, silkwormcells, and Xenopus oocyte.

When prokaryotic cells are used as host cells, an expression vector usedherein contains an origin replicable within prokaryotic cells, apromoter, a ribosome-binding site, a multiple cloning site, aterminator, a drug resistance gene, an auxotrophic complementary gene,and the like. Examples of Escherichia coli expression vector include apUC-based vector, pBluescript II, a pET expression system, and a pGEXexpression system. DNA encoding the above polypeptide is incorporatedinto such an expression vector, prokaryotic host cells are transformedwith the vector, the thus obtained transformed cells are cultured, andthus the polypeptide encoded by the DNA can be expressed in prokaryotichost cells. At this time, the polypeptide can also be expressed as afusion protein with another protein.

When eukaryotic cells are used as host cells, an expression vector usedherein is an expression vector for eukaryotic cells, which contains apromoter, a splicing region, a poly(A) addition site, and the like.Examples of such an expression vector include pKA1, pCDM8, pSVK3, pMSG,pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, and pYES2. In a mannersimilar to the above, DNA encoding the above polypeptide is incorporatedinto such an expression vector, eukaryotic host cells are transformedwith the vector, the thus obtained transformed cells are cultured, andthus the polypeptide encoded by the DNA can be expressed in eukaryotichost cells. When pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, or thelike is used as an expression vector, the above polypeptide can beexpressed as a fusion protein to which a tag from among various tagssuch as a His tag (e.g., (His)₆-(His)₁₀), a FLAG tag, a myc tag, an HAtag, and GFP has been added.

For introduction of an expression vector into host cells, a known methodcan be employed, such as electroporation, a calcium phosphate method, aliposome method, a DEAE dextran method, microinjection, viral infection,lipofection, and binding to a cell membrane-permeable peptide.

The polypeptide of interest can be isolated and purified from host cellsby a combination of known separation procedures. Examples of suchprocedures include, but are not limited to, treatment with a denaturingagent such as urea or a surfactant, ultrasonication, enzymaticdigestion, salting-out or solvent fractionation and precipitation,dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE,isoelectric focusing, ion exchange chromatography, hydrophobicchromatography, affinity chromatography, and reverse phasechromatography.

<Antibody Structure>

An antibody is a heteromultimeric glycoprotein that generally containsat least two heavy chains and two light chains. Antibodies other thanIgM is an about 150-kDa hetero tetramer glycoprotein composed of twoidentical light (L) chains and two identical heavy (H) chains.Typically, each light chain is connected to a heavy chain via onedisulfide covalent bond, however, the number of disulfide bonds betweenheavy chains of various immunoglobulin isotypes is varied. Each heavychain or each light chain also has an intrachain disulfide bond. Eachheavy chain has a variable domain (VH region) on one end followed byseveral constant regions. Each light chain has a variable domain (VLregion) and has one constant region on an end opposite to the other end.The constant region of a light chain is aligned with the first constantregion of a heavy chain, and a light chain variable domain is alignedwith a heavy chain variable domain. A specific region of an antibodyvariable domain exhibits specific variability that is referred to as acomplementarity determining region (CDR), so that it imparts bindingspecificity to the antibody. A portion of a variable region, which isrelatively conserved, is referred to as a framework region (FR).Complete heavy chain and light chain variable domains separatelycontains four FRs ligated via three CDRs. The three CDRs in a heavychain are referred to as CDRH1, CDRH2, and CDRH3 in this order from theN-terminus. Similarly, in the case of a light chain, CDRLs are referredto as CDRL1, CDRL2, and CDRL3. CDRH3 is most important for the bindingspecificity of an antibody to an antigen. Also, the CDRs of each chainare retained together in a state of being adjacent to each other due tothe FR regions, contributing to the formation of the antigen bindingsite of the antibody together with CDRs from the other chain. A constantregion does not directly contribute to the binding of an antibody to anantigen, but exhibits various effector functions, such as involvement inantibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis viabinding to an Fcγ receptor, the rate of half-life/clearance via aneonate Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC)via a C1q constituent of the complement cascade.

<Preparation of Antibody>

The term “anti-CAPRIN-1 antibody” as used herein refers to an antibodyhaving immunological reactivity with a full-length CAPRIN-1 protein or afragment thereof.

As used herein, the term “immunological reactivity” refers to theproperty of in vivo binding of an antibody to a CAPRIN-1 antigen.Through such an in vivo binding, the function of damaging tumor (e.g.,death, suppression, or degeneration) is exhibited. Specifically, anantibody used in the present invention may be any type of antibody, aslong as it binds to a CAPRIN-1 protein so as to be able to damage tumor,such as leukemia, lymphoma, breast cancer, brain tumor, lung cancer,bladder cancer, uterine cervix cancer, esophageal cancer, gastriccancer, renal cancer, or colorectal cancer.

In the present invention, examples of an antibody include, but are notparticularly limited to, as long as it is a monoclonal antibody, asynthetic antibody, a multispecific antibody, a human antibody, ahumanized antibody, a chimeric antibody, a single chain antibody, and anantibody fragment (e.g., Fab and F(ab′)₂). Also, an antibody may be animmunoglobulin molecule of any class such as IgG, IgE, IgM, IgA, IgD,and IgY, or any subclass such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The antibody may further be modified by, in addition to glycosylation,acetylation, formylation, amidation, phosphorylation, pegylation (PEG),or the like.

Preparation examples of various monoclonal antibodies are as describedbelow.

When the antibody is a monoclonal antibody, for example, the breastcancer cell line SK-BR-3 expressing CAPRIN-1 is administered to a mousefor immunization, the spleen is removed from the mouse, cells areseparated, and then the cells and mouse myeloma cells are fused. Fromamong the thus obtained fusion cells (hybridomas), a clone producing anantibody having the effect of suppressing cancer cell proliferation isselected. A hybridoma producing a monoclonal antibody that has theeffect of suppressing cancer cell proliferation is isolated, thehybridoma is cultured, and then an antibody is purified from the culturesupernatant by general affinity purification, so that the antibody canbe prepared.

The hybridoma producing a monoclonal antibody can also be prepared asdescribed below, for example. First, an animal is immunized with asensitizing antigen according to a known method. A general method iscarried out by injecting a sensitizing antigen to a mammalintraperitoneally or subcutaneously. Specifically, a sensitizing antigenis diluted with PBS (Phosphate-Buffered Saline), saline, or the like toan appropriate amount, followed by suspension. The resultant is thenmixed with an appropriate amount of a general adjuvant as necessary,such as Freund's complete adjuvant. After emulsification, the solutionwas administered to a mammal several times every 4 to 21 days.Furthermore, an appropriate carrier can also be used upon immunizationwith a sensitizing antigen.

A mammal is immunized as described above. After confirmation of a risein a desired serum antibody level, immunized cells are collected fromthe mammal and then subjected to cell fusion. Preferable immunized cellsare particularly splenocytes.

Mammalian myeloma cells are used as the other parent cells to be fusedwith the immunized cells. As the myeloma cells, various known cell linesare preferably used, such as P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8. 653) (J.Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics inMicrobiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. andMilstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D.H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature(1978) 276, 269-270), FO (deSt. Groth, S. F. et al., J. Immunol. Methods(1980) 35, 1-21), 5194 (Trowbridge, I. S. J. Exp. Med. (1978) 148,313-323), and 8210 (Galfre, G. et al., Nature (1979) 277, 131-133).

Fusion of the immunized cell and the myeloma cell can be carried outaccording to basically a known method such as Kohler and Milstein'stechnique (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73,3-46), for example.

More specifically, the above cell fusion is carried out, for example, inthe presence of a cell fusion accelerator in a usual nutrient culturemedium. As this fusion accelerator, polyethyleneglycol (PEG), Sendaivirus (HVJ), or the like is used. If desired, an auxiliary agent such asdimethylsulfoxide may be added and used in order to enhance fusionefficiency.

The ratio of the immunized cells to the myeloma cells to be used hereincan be arbitrarily set. For example, the number of immunized cells thatare preferably used is one to ten times the number of myeloma cells. Asa culture medium to be used for the above-mentioned cell fusion, anRPMI1640 culture medium suitable for proliferation of theabove-mentioned myeloma cell line, an MEM culture medium, and otherculture media usually used for culturing this kind of cell can be used.Further, liquid that is supplemental to serum such as fetal bovine serum(FCS) can be used together therewith.

Cell fusion can be performed by thoroughly mixing the predeterminedamounts of the above immunized cells and the myeloma cells in the aboveculture medium, and a PEG solution (for example, having an averagemolecular weight ranging from about 1000 to 6000) prewarmed at about 37°C. is added usually at a concentration of 30%-60% (w/v) and mixed,thereby forming a culture containing hybridomas of interest. Next, asuitable culture medium is successively added to the thus-obtainedculture, which is then centrifuged to remove the supernatant, and thisprocedure is repeated to remove the cell fusion agent or the like whichis not preferable for the growth of hybridomas.

The thus obtained hybridomas are cultured for selection in a usualselection culture medium (e.g., a HAT culture medium containinghypoxanthine, aminopterin and thymidine). Culturing in this HAT culturemedium is continued for a sufficient period of time (usually severaldays to several weeks) so that the cells (non-fused cells) other thanthe target hybridomas die. Subsequently, screening and single cloning ofthe hybridoma which produces an antibody of interest are performed usingthe general limiting dilution method.

The above hybridomas are obtained by an immunizing non-human animal withan antigen. In addition to this method, hybridomas that produce a humanantibody having desired activity (e.g., activity of suppressing cellproliferation) can also be obtained by in vitro sensitizing humanlymphocytes, such as human lymphocytes that have been infected with theEB virus, with a protein, a protein-expressing cell, or a lysatethereof, followed by fusing of the thus sensitized lymphocytes withhuman-derived myeloma cells having an ability to permanently divide,such as U266 (Accession No. TIB196).

The thus prepared hybridoma that produces a monoclonal antibody ofinterest can be passaged in a general culture medium and can be storedin liquid nitrogen over a long period of time.

Specifically, a hybridoma can be prepared by immunizing by a generalimmunization method using, as a sensitizing antigen, a desired antigenor a cell that expresses the desired antigen, fusing the thus obtainedimmunized cell with a known parent cell by a general cell fusion method,and then screening for a monoclonal antibody-producing cell (i.e., ahybridoma) by a general screening method.

As a human antibody-producing mouse, a KM mouse (Kirin Pharma/Medarex)and a Xeno mouse (Amgen) are known (e.g., International PatentPublications WO02/43478 and WO02/092812), for example. When such a mouseis immunized with a CAPRIN-1 protein or a fragment thereof, a completehuman polyclonal antibody can be obtained from blood. Also, splenocytesare collected from the immunized mouse and then a human-type monoclonalantibody can be prepared by a method for fusion with myeloma cells.

An antigen can be prepared according to a method using animal cells (JPPatent Publication (Kohyo) No. 2007-530068) or baculovirus (e.g.,International Patent Publication WO98/46777), for example. When anantigen has low immunogenicity, the antigen may be bound to amacromolecule having immunogenicity, such as albumin, and thenimmunization is carried out.

Furthermore, an antibody gene is cloned from said hybridoma and thenincorporated into an appropriate vector. The vector is then introducedinto a host, and then the genetically recombined antibody produced usinggene recombination techniques can be used (e.g., see Carl, A. K.Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).Specifically, the cDNA of a variable region (V region) of an antibody issynthesized from the mRNA of the hybridoma using reverse transcriptase.When DNA encoding the V region of an antibody of interest can beobtained, this DNA is ligated to DNA encoding the constant region (Cregion) of a desired antibody, and then the resultant fusion product isincorporated into an expression vector. Alternatively, DNA encoding theV region of an antibody may be incorporated into an expression vectorcontaining the DNA for the C region of an antibody. At this time, theDNA can be incorporated into an expression vector so that it isexpressed under the control of expression control regions, such asenhancer and promoter. Next, host cells are transformed with theexpression vector, so that the antibody can be expressed.

The anti-CAPRIN-1 antibody of the present invention is characterized bybeing a monoclonal antibody. Examples of a monoclonal antibody includehuman monoclonal antibodies, non-human animal monoclonal antibodies(e.g., a mouse monoclonal antibody, a rat monoclonal antibody, a rabbitmonoclonal antibody, and a chicken monoclonal antibody), and chimericmonoclonal antibodies. A monoclonal antibody can be prepared byculturing a hybridoma obtained by cell fusion of a splenocyte from anon-human mammal (e.g., a mouse, a human antibody-producing mouse, achicken, or a rabbit) immunized with a CAPRIN-1 protein, with a myelomacell. A chimeric antibody is prepared by combining sequences fromdifferent animals, such as an antibody comprising heavy chain and lightchain variable regions of a mouse antibody and heavy chain and lightchain constant regions of a human antibody. A chimeric antibody can beprepared using a known method. For example, a chimeric antibody can beobtained by ligating DNA encoding an antibody V region to DNA encoding ahuman antibody C region, incorporating the resultant fusion product intoan expression vector, and then introducing the vector into a host forproduction of the chimeric antibody. In Examples described later,human-chicken chimeric monoclonal antibodies were prepared and thus itsanti-tumor effects were confirmed. These monoclonal antibodies comprisea heavy chain variable (VH) region comprising the amino acid sequence ofSEQ ID NO: 42 and a light chain variable (VL) region comprising theamino acid sequence of SEQ ID NO: 46, wherein the VH region comprisesCDR1 represented by the amino acid sequence of SEQ ID NO: 39, CDR2represented by the amino acid sequence of SEQ ID NO: 40, and CDR3represented by the amino acid sequence of SEQ ID NO: 41, and the VLregion comprises CDR1 represented by the amino acid sequence of SEQ IDNO: 43, CDR2 represented by the amino acid sequence of SEQ ID NO: 44,and CDR3 represented by the amino acid sequence of SEQ ID NO: 45.

A humanized antibody is a modified antibody that is also referred to asa reshaped human antibody. A humanized antibody can be constructed bytransplanting CDRs of an antibody from an immunized animal into thecomplementarity determining regions of a human antibody. General generecombination techniques therefor are also known.

Specifically, DNA sequences designed to have each of the CDRs of a mouseor chicken antibody ligated to each of the framework regions (FRs) of ahuman antibody are synthesized by the PCR method from severaloligonucleotides, which are prepared so as to have overlap portions attheir terminal portions, for example. A humanized antibody can beobtained by ligating the thus obtained DNA to DNA encoding the constantregion of a human antibody, incorporating the resultant fusion productinto an expression vector, introducing the vector into a host, and thuscausing the host to produce the gene product (see European PatentPublication No. 239400 and International Patent Publication WO96/02576).As the FRs of a human antibody, which is ligated via CDRs, FRs thatallow the formation of an antigen-binding site with good complementaritydetermining regions are selected. If necessary, for the formation of anantigen-binding site having the appropriate complementarity determiningregions of a reshaped human antibody, the amino acids of the frameworkregions of an antibody variable region may be substituted (Sato, K. etal., Cancer Research, 1993, 53: 851-856). Also, the amino acids of FRsmay be substituted with those of framework regions from various humanantibodies (see International Patent Publication WO99/51743).

As the framework regions (FRs) of a human antibody, which are ligatedvia CDRs, FRs that allows the formation of an antigen-binding site withgood complementarity determining regions are selected. If necessary, forthe formation of an antigen-binding site having the appropriatecomplementarity determining regions of a reshaped human antibody, theamino acids of the framework regions of an antibody variable region maybe substituted (Sato K. et al., Cancer Research 1993, 53: 851-856).

After preparation of a chimeric antibody or a humanized antibody, aminoacids in a variable region (e.g., FR) or a constant region may besubstituted with other amino acids.

Amino acid substitution is a substitution of, for example, less than 15,less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3or less, or 2 or less amino acids and is preferably a substitution of 1to 5 amino acids, and more preferably 1 or 2 amino acids. A substitutedantibody should be functionally equivalent to an unsubstituted antibody.Substitution is desirably a substitution of a conservative amino acid(s)between amino acids having analogous properties such as electric charge,side chain, polarity, and aromaticity. Amino acids having analogousproperties can be classified into basic amino acids (arginine, lysine,and histidine), acidic amino acids (aspartic acid and glutamic acid),uncharged polar amino acids (glycine, asparagine, glutamine, serine,threonine, cysteine, and tyrosine), nonpolar amino acids (leucine,isoleucine, alanine, valine, proline, phenylalanine, tryptophan, andmethionine), branched-chain amino acids (threonine, valine, andisoleucine), and aromatic amino acids (phenylalanine, tyrosine,tryptophan, and histidine), for example.

Examples of a modified antibody product include antibodies bound tovarious molecules such as polyethylene glycol (PEG). Substances to bebound in the modified antibody product of the present invention are notlimited. Such a modified antibody product can be obtained by subjectingthe thus obtained antibody to chemical modification. Methods thereforhave already been established in the art.

As used herein, the term “functionally equivalent” refers to that asubject antibody has biological or biochemical activity similar to thatof the antibody of the present invention, and specifically refers tothat a subject antibody has the function of impairing tumor withoutessentially causing rejection upon its application to a human, forexample. An example of such activity includes an activity to suppresscell proliferation or a binding activity.

As a method well known by persons skilled in the art for preparation ofa polypeptide functionally equivalent to a polypeptide, a method forintroducing a mutation into a polypeptide is known. For example, personsskilled in the art can prepare an antibody functionally equivalent tothe antibody of the present invention by appropriately introducing amutation into the antibody using site-directed mutagenesis(Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275; Zoller, M J., andSmith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al.,(1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, H J.,(1987) Methods Enzymol. 154, 350-367; Kunkel, T A., (1985) Proc. Natl.Acad. Sci. U.S.A. 82, 488-492; Kunkel (1988) Methods Enzymol. 85,2763-2766), for example.

An antibody that recognizes an epitope of a CAPRIN-1 protein recognizedby the above anti-CAPRIN-1 antibody can be obtained by a method known bypersons skilled in the art. For example, such an antibody can beobtained by a method that involves determining an epitope of a CAPRIN-1protein recognized by an anti-CAPRIN-1 antibody, by a general method(e.g., epitope mapping) and then preparing an antibody using apolypeptide having an amino acid sequence contained in the epitope as animmunogen, or a method that involves determining an epitope of such anantibody prepared by a general method, and then selecting an antibodyhaving the epitope identical with that of an anti-CAPRIN-1 antibody. Asused herein, the term “epitope” refers to, in a mammal and preferably ahuman, a polypeptide fragment having antigenicity or immunogenicity. Theminimum size unit thereof consists of about 7 to 12 amino acids, andpreferably 8 to 11 amino acids.

The affinity constant Ka(k_(on)/k_(off)) of the antibody of the presentinvention is preferably at least 10⁷ M⁻¹, at least 10⁸ M⁻¹, at least5×10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, atleast 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹²M⁻¹, or at least 10¹³ M⁻¹.

The antibody of the present invention can be conjugated with anantitumor agent. Conjugation of the antibody with an antitumor agent canbe carried out via a spacer having a group reactive with an amino group,a carboxyl group, a hydroxy group, a thiol group or the like (e.g., asuccinimidyl succinate group, a formyl group, a 2-pyridyldithio group, amaleimidyl group, an alkoxy carbonyl group, and a hydroxy group).

Examples of the antitumor agent include the following known antitumoragents as in prior art literatures and the like, such as paclitaxel,doxorubicin, daunorubicin, cyclophosphamide, methotrexate,5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa,carboquone, meturedopa, uredopa, altretamine, triethylenemelamine,triethylenephosphoramide, triethilenethiophosphoramide,trimethylolomelamine, bullatacin, bullatacinone, camptothecin,bryostatin, callystatin, cryptophycin1, cryptophycin8, dolastatin,duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin,chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard,carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine, calicheamicin, dynemicin, clodronate, esperamicin,aclacinomycin, actinomycin, authramycin, azaserine, bleomycin,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin,dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine, adriamycin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycinC,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin,trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine,ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens(e.g., calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone), aminoglutethimide, mitotane,trilostane, frolinic acid, aceglatone, aldophosphamideglycoside,aminolaevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene,edatraxate, defofamine, demecolcine, diaziquone, elformithine,elliptinium acetate, epothilone, etoglucid, lenthinan, lonidamine,maytansine, ansamitocine, mitoguazone, mitoxantrone, mopidanmol,nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone,podophyllinic acid, 2-ethyl hydrazide, procarbazine, razoxane, rhizoxin,schizophyllan, spirogermanium, tenuazonic acid, triaziquone, roridine A,anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol,mitolactol, pipobroman, gacytosine, docetaxel, chlorambucil,gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, oxaliplatin,carboplatin, vinblastine, etoposide, ifosfamide, mitoxanthrone,vincristine, vinorelbine, novantrone, teniposide, edatrexate,daunomycin, aminopterin, xeloda, ibandronate, irinotecan, topoisomeraseinhibitor, difluoromethylolnitine (DMFO), retinoic acid, capecitabine,and pharmaceutically acceptable salts or derivatives thereof.

Through administration of the antibody of the present invention incombination with an antitumor agent, even higher therapeutic effects canbe obtained. This technique is applicable to both before and aftersurgery of a cancer patient with the expression of CAPRIN-1.Particularly after surgery, more effective prevention of cancerrecurrences or prolonged survival period can be obtained against cancerwith the expression of CAPRIN-1, which has been conventionally treatedwith an antitumor agent alone.

Examples of the antitumor agent to be administered in combination withthe antibody of the present invention include the following knownantitumor agents as in prior art literatures or the like, such aspaclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate,5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, benzodopa,carboquone, meturedopa, uredopa, altretamine, triethylenemelamine,triethylenephosphoramide, triethilenethiophosphoramide,trimethylolomelamine, bullatacin, bullatacinone, camptothecin,bryostatin, callystatin, cryptophycin1, cryptophycin8, dolastatin,duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin,chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine, calicheamicin, dynemicin, clodronate,esperamicin, aclacinomycin, actinomycin, authramycin, azaserine,bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin,chromomycin, dactinomycin, detorbicin, 6-diazo-5-oxo-L-norleucine,adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin,mitomycinC, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin,pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine,thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid,aceglatone, aldophosphamideglycoside, aminolaevulinic acid, eniluracil,amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine,diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid,lenthinan, lonidamine, maytansine, ansamitocine, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethyl hydrazide,procarbazine, razoxane, rhizoxin, schizophyllan, spirogermanium,tenuazonic acid, triaziquone, roridine A, anguidine, urethane,vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol,pipobroman, gacytosine, docetaxel, chlorambucil, gemcitabine,6-thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin,vinblastine, etoposide, ifosfamide, mitoxanthrone, vincristine,vinorelbine, novantrone, teniposide, edatrexate, daunomycin,aminopterin, xeloda, ibandronate, irinotecan, topoisomerase inhibitor,difluoromethylolnitine (DMFO), retinoic acid, capecitabine, andpharmaceutically acceptable (known) salts or (known) derivativesthereof. Of the above examples, particularly cyclophosphamide,paclitaxel, docetaxel, and vinorelbine are preferably used.

Alternatively, a known radio isotope as in prior art literatures or thelike, such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P,¹⁷⁵Lu, or ¹⁷⁶Lu can be bound to the antibody of the present invention. Adesired radio isotope is effective for treatment or diagnosis of tumor.

The antibody of the present invention is an antibody havingimmunological reactivity with CAPRIN-1, or, an antibody specificallybinding to CAPRIN-1, which exhibits cytotoxic activity against cancer orthe effect of suppressing tumor growth. The antibody should have astructure such that rejection is almost or completely avoided in asubject animal to which the antibody is administered. Examples of suchan antibody include, when a subject animal is a human, a human antibody,a humanized antibody, a chimeric antibody (e.g., a human-mouse chimericantibody), a single chain antibody, and a bispecific antibody. Theseantibodies are: recombinant antibodies in which heavy chain and lightchain variable regions are from a human antibody; recombinant antibodiesin which heavy chain and light chain variable regions comprisecomplementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) from anon-human animal antibody and framework regions from a human antibody;or recombinant antibodies in which heavy chain and light chain variableregions are from a non-human animal antibody, and, heavy chain and lightchain constant regions are from a human antibody. Preferable antibodiesare the former two antibodies.

These recombinant antibodies can be prepared as follows by cloning DNAencoding an anti-human CAPRIN-1 monoclonal antibody (e.g., a humanmonoclonal antibody, a mouse monoclonal antibody, a rat monoclonalantibody, a rabbit monoclonal antibody, or a chicken monoclonalantibody) from an antibody-producing cell such as a hybridoma, preparingDNA encoding a light chain variable region and a heavy chain variableregion of the antibody by an RT-PCR method using it as a template, andthen determining the sequence of each variable region of light chain andheavy chain or each sequence of CDR1, CDR2, and CDR3 based on a Kabat EUnumbering system (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed. Public Health Service, National Institute ofHealth, Bethesda, Md. (1991)).

Furthermore, DNA encoding each of these variable regions or DNA encodingeach CDR is prepared using gene recombination techniques (Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989)) or a DNA synthesizer. Here, the above humanmonoclonal antibody-producing hybridoma can be prepared by immunizing ahuman antibody-producing animal (e.g., a mouse) with human CAPRIN-1 andthen fusing splenocytes excised from the immunized animal to myelomacells. Alternatively, DNAs encoding a light chain or heavy chainvariable region and a constant region from a human antibody are preparedas necessary using gene recombination techniques or a DNA synthesizer.

In the case of humanized antibody, DNA is prepared by substituting a CDRcoding sequence in DNA encoding a variable region of light chain orheavy chain derived from a human antibody, with a CDR coding sequencecorresponding thereto of an antibody derived from a non-human animal(e.g., a mouse, a rat, or a chicken) and then ligating the DNA thusobtained to DNA encoding a constant region of light chain or heavy chainderived from a human antibody. Thus, DNA encoding humanized antibody canbe prepared.

In the case of chimeric antibody, DNA encoding a chimeric antibody canbe prepared by ligating DNA encoding a light chain or heavy chainvariable region of an antibody from a non-human animal (e.g., a mouse, arat, and a chicken) to DNA encoding a light chain or heavy chainconstant region from a human antibody.

In the case of single chain antibody, this antibody is an antibodyprepared by linearly ligating a heavy chain variable region to a lightchain variable region via a linker. Thus, DNA encoding a single chainantibody can be prepared by binding DNA encoding a heavy chain variableregion, DNA encoding a linker, and DNA encoding a light chain variableregion. Herein, a heavy chain variable region and a light chain variableregion are both from a human antibody, or, only CDRs are substitutedwith CDRs of an antibody from a non-human animal (e.g., a mouse, a rat,and a chicken) although the other regions are from a human antibody.Also, a linker comprises 12 to 19 amino acids, such as (G₄S)₃ of 15amino acids (G.-B. Kim et al., Protein Engineering Design and Selection2007, 20 (9): 425-432).

In the case of bispecific antibody (diabody), this antibody is capableof specifically binding to two different epitopes. For example, DNAencoding a bispecific antibody can be prepared by linking DNA encoding aheavy chain variable region A, DNA encoding a light chain variableregion B, DNA encoding a heavy chain variable region B, and DNA encodinga light chain variable region A in this order (here, DNA encoding alight chain variable region B is bound to DNA encoding a heavy chainvariable region B via DNA encoding the above linker). Here, a heavychain variable region and a light chain variable region are both from ahuman antibody, or, only CDRs are substituted with CDRs of an antibodyfrom a non-human animal (e.g., a mouse, a rat, or a chicken) althoughthe other regions are from a human antibody.

The above-prepared recombinant DNA is incorporated into one or aplurality of appropriate vectors, they are introduced into host cells(e.g., mammalian cells, yeast cells, or insect cells), and then(co)expression is caused, so that a recombinant antibody can be prepared(P. J. Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY,P. Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITYPRESS; J. W. Goding., Monoclonal Antibodies: principles and practice.,1993 ACADEMIC PRESS).

Examples of the antibody of the present invention prepared by the abovemethod include an antibody comprising a heavy chain variable regioncomprising SEQ ID NOS: 39, 40, and 41 and a light chain variable regioncomprising SEQ ID NOS: 43, 44, and 45 (e.g., the antibody composed ofthe heavy chain variable region of SEQ ID NO: 42 and the light chainvariable region of SEQ ID NO: 46).

The amino acid sequences represented by SEQ ID NOS: 39, 40, and 41 areCDR1, CDR2, and CDR3 of a chicken antibody heavy chain variable region.Also, the amino acid sequences represented by SEQ ID NOS: 43, 44, and 45are CDR1, CDR2, and CDR3 of a chicken antibody light chain variableregion, respectively.

Also, the humanized antibody, the chimeric antibody, the single chainantibody, or the bispecific antibody of the present invention is thefollowing antibody, for example:

(i) an antibody wherein a heavy chain variable region comprises theamino acid sequences of SEQ ID NOS: 39, 40, and 41 and the amino acidsequences of framework regions from a human antibody, and, a light chainvariable region comprises the amino acid sequences of SEQ ID NOS: 43,44, and 45 and the amino acid sequences of framework regions from ahuman antibody (e.g., the antibody wherein the heavy chain variableregion comprises the amino acid sequence of SEQ ID NO: 42, and, thelight chain variable region comprises the amino acid sequence of SEQ IDNO: 46); and(ii) an antibody wherein a heavy chain variable region comprises theamino acid sequences of SEQ ID NOS: 39, 40, and 41 and the amino acidsequences of framework regions from a human antibody, and, a heavy chainconstant region comprises an amino acid sequence from a human antibody,and, a light chain variable region comprises the amino acid sequences ofSEQ ID NOS: 43, 44, and 45 and the amino acid sequences of frameworkregions from a human antibody, and a light chain constant regioncomprises an amino acid sequence from a human antibody (e.g., theantibody wherein a heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 42, and, a heavy chain constant region comprisesan amino acid sequence from a human antibody, as well as, a light chainvariable region comprises the amino acid sequence of SEQ ID NO: 46, and,a light chain constant region comprises an amino acid sequence from ahuman antibody).

In addition, the sequences of human antibody heavy chain and light chainconstant regions and variable regions can be obtained from NCBI (e.g.,U.S.A.: GenBank, UniGene), for example. For example, the sequence ofAccession No. J00228 can be referred to as a human IgG1 heavy chainconstant region, the sequence of Accession No. J00230 can be referred toas a human IgG2 heavy chain constant region, the sequence of AccessionNo. X03604 can be referred to as a human IgG3 heavy chain constantregion, the sequence of Accession No. K01316 can be referred to as ahuman IgG4 heavy chain constant region, the sequences of Accession No.V00557, X64135, X64133, and the like can be referred to as human lightchain κ constant regions, and the sequences of registration nos. X64132,X64134, and the like can be referred to as human light chain λ constantregions.

The above antibodies preferably have cytotoxic activity and thus canexhibit anti-tumor effects.

Also, the specific sequences of heavy chain and light chain variableregions or CDRs in the above antibodies are given simply forillustrative purposes, and thus are clearly not limited to such specificsequences. A hybridoma capable of producing another human antibody ornon-human animal antibody (e.g., a mouse antibody) against humanCAPRIN-1 is prepared, a monoclonal antibody that is produced by thehybridoma is collected, and then whether or not it is a target antibodyis determined by immunological binding property with human CAPRIN-1 andcytotoxic activity as indicators. After identification of a hybridomaproducing the target monoclonal antibody in this manner, DNA encodingheavy chain and light chain variable regions of the target antibody isprepared from the hybridoma as described above, sequencing is carriedout, and then the DNA is used for preparation of another antibody.

Furthermore, the above antibody of the present invention, the sequenceof each of the above antibodies, particularly the sequence of theframework region and/or the sequence of the constant region may have asubstitution, a deletion, or an addition of one or several (preferably,1 or 2) amino acids, as long as it has specificity for specificrecognition of CAPRIN-1. Here the term “several” refers to 2 to 5, andpreferably 2 or 3.

The present invention further provides DNA encoding the above antibodyof the present invention, or, DNA encoding the above antibody heavychain or light chain, or, DNA encoding the above antibody heavy chain orlight chain variable region. Examples of such DNA include, DNA encodinga heavy chain variable region comprising the nucleotide sequencesencoding the amino acid sequences of SEQ ID NOS: 39, 40, and 41 and DNAencoding a light chain variable region comprising the nucleotidesequences encoding the amino acid sequences of SEQ ID NOS: 43, 44, and45.

Complementarity determining regions (CDRs) encoded by the sequences ofDNA are regions for determining the specificity of an antibody. Thus,sequences encoding regions in an antibody other than CDRs (specifically,a constant region and a framework region) may be from other antibodies.Here, examples of such “other antibodies” include antibodies fromnon-human organisms, and are preferably from a human in view ofreduction of side effects. Thus, in the case of the above DNA, regionsencoding each framework region and each contact region of heavy chainsand light chains preferably comprise nucleotide sequences encodingcorresponding amino acid sequences from a human antibody.

Further alternative examples of DNA encoding the antibody of the presentinvention include DNA encoding a heavy chain variable region comprisingthe nucleotide sequence encoding the amino acid sequence of SEQ ID NO:42 and DNA in which the region encoding a light chain variable regioncomprises the nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 46. Here, an example of the nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 42 is the nucleotide sequence of SEQID NO: 49. Also, an example of the nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 46 is the nucleotide sequence of SEQID NO: 50. In these DNAs, regions encoding each constant region of heavychains and light chains preferably comprise nucleotide sequencesencoding the corresponding amino acid sequences from a human antibody.

The DNA of the present invention can be obtained by the above methods orthe following method, for example. First, total RNA is prepared from ahybridoma relating to the antibody of the present invention using acommercially available RNA extraction kit, and then cDNA is synthesizedwith reverse transcriptase using random primers, and the like.Subsequently, cDNA encoding an antibody is amplified by a PCR methodusing as primers the oligonucleotides of sequences conserved in eachvariable region of known mouse antibody heavy chain and light chaingenes. The sequence encoding a constant region can be obtained byamplifying a known sequence by a PCR method. The nucleotide sequence ofDNA can be determined by a conventional method such as insertion of itinto a plasmid or a phage for sequencing.

An anti-CAPRIN-1 antibody to be used in the present invention isconsidered to exhibit the anti-tumor effects against CAPRIN-1-expressingcancer cells through the following mechanism.

Antibody-dependent cytotoxic activity (ADCC) of effector cells againstCAPRIN-1-expressing cells, and the complement-dependent cytotoxicity(CDC) against CAPRIN-1-expressing cells.

Therefore, the activity of an anti-CAPRIN-1 antibody to be used in thepresent invention can be evaluated by, as specifically described inExamples below, measuring ex vivo the above ADCC activity or CDCactivity against CAPRIN-1-expressing cancer cells.

An anti-CAPRIN-1 antibody to be used in the present invention binds to aCAPRIN-1 protein on a cancer cell and exhibits anti-tumor effects due tothe above activity, and thus it is useful for treating or preventingcancer. Specifically, the present invention provides a pharmaceuticalcomposition for treating and/or preventing a cancer, which comprises ananti-CAPRIN-1 antibody as an active ingredient. When the anti-CAPRIN-1antibody is used for administration thereof to a human body (antibodytherapy), it is preferably human antibody or humanized antibody in orderto decrease immunogenicity.

In addition, the higher the binding affinity between an anti-CAPRIN-1antibody and a CAPRIN-1 protein on the cancer cell surfaces, thestronger the anti-tumor activity of the anti-CAPRIN-1 antibody that canbe obtained. Therefore, when an anti-CAPRIN-1 antibody having highbinding affinity with a CAPRIN-1 protein can be acquired, strongeranti-tumor effects can be expected and such antibody's application as apharmaceutical composition for the purpose of cancer treatment and/orprevention becomes possible. Such high binding affinity is desirably asfollows. As described above, binding constant (affinity constant) Ka(k_(on)/k_(off)) is preferably at least 10⁷ M⁻¹, at least 10⁸ M⁻¹, atleast 5×10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, atleast 10¹² M⁻¹, or, at least 10¹³ M⁻¹.

<Binding to Antigen-Expressing Cell>

The capacity of an antibody to bind to CAPRIN-1 can be specified bybinding assay using ELISA, a Western blot method, immuno-fluorescenceand flow cytometric analysis, or the like as described in Examples.

<Immunohistochemical Staining>

An antibody that recognizes CAPRIN-1 can be tested for reactivity toCAPRIN-1 by a method for immunohistochemistry known by persons skilledin the art using paraformaldehyde- or acetone-fixed frozen sections orparaformaldehyde-fixed paraffin-embedded tissue sections, which isprepared from tissue samples obtained from a patient during surgery, ortissue samples obtained from an animal having heterotransplant tissueinoculated with a cell line expressing CAPRIN-1, naturally or aftertransfection.

An antibody reactive with CAPRIN-1 can be stained by various methods forimmunohistochemical staining. For example, a horseradishperoxidase-conjugated goat anti-mouse antibody or goat anti-chickenantibody is caused to undergo reaction, a target antibody can bevisualized.

<Pharmaceutical Composition>

A target of the pharmaceutical composition for treating and/orpreventing a cancer of the present invention is not particularlylimited, as long as it is a cancer (cell) expressing a CAPRIN-1 gene.

The term “tumor” and “cancer” as used herein refers to malignantneoplasm and is used interchangeably.

Cancer to be subjected to the present invention is cancer expressinggenes encoding CAPRIN-1 proteins having amino acid sequences ofeven-numbered SEQ ID NOS: 2 to 30. Examples of such cancer includepreferably breast cancer, brain tumor, leukemia, lung cancer, lymphoma,mastocytoma, renal cancer, uterine cervix cancer, bladder cancer,esophageal cancer, gastric cancer, and colorectal cancer.

Examples of such specific cancer include, but are not limited to, breastadenocarcinoma, composite type breast adenocarcinoma, mammary glandmalignant mixed tumor, intraductal papillary adenocarcinoma, lungadenocarcinoma, squamous cell carcinoma, small cell carcinoma, largecell carcinoma, glioma that is neural epithelial tissue tumor,ependymoma, neurocytoma, fetal neuroectodermal tumor, schwannoma,neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma,gastrointestinal lymphoma, digestive lymphoma, small-cell to medium-celllymphoma, cancer of cecum, ascending colon cancer, descending coloncancer, transverse colon cancer, sigmoid colon cancer, and rectalcancer.

Moreover, preferable subjects are mammals including primates, petanimals, domestic animals, animals for race, and the like and areparticularly preferably humans, dogs, and cats.

When an antibody to be used in the present invention is used as apharmaceutical composition, it can be formulated by a method known bypersons skilled in the art. For example, the antibody can be usedparenterally in the form of an injection preparation such as an asepticsolution or a suspension prepared with water or a pharmacologicallyacceptable solution other than water. For example, it can be formulatedby mixing in a unit dosage form required by generally acceptedpharmaceutical practice in appropriate combination with apharmacologically acceptable carrier or medium, specifically, sterilewater or saline, vegetable oil, an emulsifier, a suspension, asurfactant, a stabilizer, a flavoring compound, an excipient, a vehicle,an antiseptic, a binder, and the like. The amounts of active ingredientsin these preparations are determined so that an appropriate dose withinthe indicated range can be obtained.

An aseptic composition for injection can be prescribed according togeneral pharmaceutical practice using a vehicle such as distilled waterfor injection.

Examples of an aqueous solution for injection include saline, anisotonic solution containing dextrose or other adjuvants, such asD-sorbitol, D-mannose, D-mannitol, and sodium chloride. These examplesmay be used in combination with an appropriate solubilizing agent suchas alcohol, specifically ethanol and polyalcohol (e.g., propylene glycoland polyethylene glycol), and nonionic surfactant (e.g., polysorbate 80(TM) and HCO-60).

Examples of the oil include sesame oil and soybean oil, which can beused in combination with a solubilizing agent such as benzyl benzoate orbenzyl alcohol. Also, a buffering agent such as phosphate buffer orsodium acetate buffer, a soothing agent such as procaine hydrochloride,a stabilizer such as benzyl alcohol or phenol, and an antioxidant may becombined therewith. An appropriate amplus is generally filled with thethus prepared injection solution.

Administration is peroral or perenteral administration and is preferablyperenteral administration. Specific examples of the route ofadministration include injection, transnasal administration, pulmonaryadministration, and transdermal administration. Examples of injectioninclude intravenous injection, intramuscular injection, intraperitonealinjection, and subcutaneous injection, so that systemic or localadministration is possible.

Also, administration methods can be appropriately selected depending ona patient's age, body weight, gender, symptoms, and the like. The dosageper administration of a pharmaceutical composition containing anantibody or a polynucleotide encoding the antibody can be selected fromthe range between 0.0001 mg and 1000 mg per kg of body weight, forexample. Alternatively, for example, dosage can be selected from therange between 0.001 mg/body and 100000 mg/body per patient. However, thedosage range is not always limited to these numerical values. The dosageand administration method are varied depending on a patient's bodyweight, age, gender, symptoms, and the like, but can be appropriatelyselected by persons skilled in the art.

The above pharmaceutical composition containing the antibody or afragment thereof of the present invention is administered to a subject,so that cancer, preferably, breast cancer, brain tumor, leukemia, lungcancer, lymphoma, mastocytoma, renal cancer, uterine cervix cancer,bladder cancer, esophageal cancer, gastric cancer, and colorectal cancercan be treated and/or prevented.

The present invention further encompasses a method for treating and/orpreventing a cancer, comprising administering to a subject thepharmaceutical composition of the present invention in combination withthe above exemplified antitumor agent or pharmaceutical compositioncontaining such antitumor agent. The antibody or a fragment thereof ofthe present invention and an antitumor agent may be administeredsimultaneously or separately to a subject. They can be separatelyadministered regardless of the order of administration. Theadministration intervals, dosage, the route of administration, and thefrequency of administration can be appropriately selected by aspecialist. Examples of the other pharmaceutical formulation to beadministered simultaneously include pharmaceutical compositions obtainedby mixing the antibody or a fragment thereof of the present inventionwith an antitumor agent in a pharmacologically acceptable carrier (or amedium) followed by formulation. Furthermore, about either the abovepharmaceutical composition containing an antitumor agent andformulation, explanations concerning prescription, formulation, theroute of administration, dose, cancer, and the like for administering apharmaceutical composition containing the antibody of the presentinvention and formulation are applicable.

Therefore, the present invention also provides a pharmaceuticalcombination for treating and/or preventing a cancer, comprising thepharmaceutical composition of the present invention, and the aboveexemplified pharmaceutical composition containing an antitumor agent.

Also, the present invention provides a pharmaceutical composition fortreating and/or preventing a cancer, comprising the antibody or afragment thereof of the present invention and an antitumor agenttogether with a pharmacologically acceptable carrier.

<Polypeptide and DNA>

The present invention further provides the following polypeptides andDNAs relating to the above antibodies.

(i) A polypeptide comprising the amino acid sequence of SEQ ID NO: 42and DNA encoding the polypeptide, wherein the DNA comprises thenucleotide sequence of SEQ ID NO: 49.(ii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 46and DNA encoding the polypeptide, wherein the DNA comprises thenucleotide sequence of SEQ ID NO: 50.(iii) A heavy chain CDR polypeptide consisting of the amino acidsequences represented by SEQ ID NOS: 39, 40, and 41, and DNA encodingthe polypeptide.(iv) A light chain CDR polypeptide consisting of the amino acidsequences represented by SEQ ID NOS: 43, 44, and 45, and DNA encodingthe polypeptide.

These polypeptides and DNAs can be prepared using gene recombinationtechniques as described above.

<Summary of the Present Invention>

The above-explained present invention is as summarized as follows.

(1) A pharmaceutical composition for treating and/or preventing acancer, comprising an antibody that comprises a heavy chain variableregion comprising SEQ ID NOS: 39, 40, and 41 and a light chain variableregion comprising SEQ ID NOS: 43, 44, and 45 or a fragment thereof as anactive ingredient and has immunological reactivity with a CAPRIN-1protein.(2) The pharmaceutical composition according to (1) above, wherein thecancer is breast cancer, brain tumor, leukemia, lymphoma, lung cancer,mastocytoma, renal cancer, uterine cervix cancer, esophageal cancer,gastric cancer, bladder cancer, or colorectal cancer.(3) The pharmaceutical composition according to (1) or (2) above,wherein the antibody is a human antibody, humanized antibody, chimericantibody, single chain antibody, or bispecific antibody.(4) An antibody, which comprises a heavy chain variable regioncomprising SEQ ID NOS: 39, 40, and 41 and a light chain variable regioncomprising SEQ ID NOS: 43, 44, and 45, and has immunological reactivitywith a CAPRIN-1 protein.(5) The antibody according to (4) above, which is a human antibody,humanized antibody, chimeric antibody, single chain antibody, orbispecific antibody.(6) A pharmaceutical combination for treating and/or preventing acancer, comprising the pharmaceutical composition of any one of (1) to(3) above and a pharmaceutical composition containing an antitumoragent.(7) A method for treating and/or preventing a cancer, comprisingadministering to a subject the pharmaceutical composition according toany one of (1) to (3) above or the antibody of any one of (3) to (5)above or a fragment thereof.(8) A method for treating and/or preventing a cancer, comprising usingpharmaceutical compositions of the pharmaceutical combination of (6)above in combination in a subject.

EXAMPLES

The present invention is described more specifically based on Examples,but the scope of the present invention is not limited by these specificexamples.

Example 1 Identification of Novel Cancer Antigen Protein by SEREX Method

(1) Preparation of cDNA Library

Total RNA was extracted from a testis tissue of a healthy dog by an acidguanidium-phenol-chloroform method. PolyA RNA was purified according toprotocols included with an Oligotex-dT30 mRNA purification Kit (TakaraShuzo Co., Ltd.) using the kit.

A dog testis cDNA phage library was synthesized using the thus obtainedmRNA (5 μg). For preparation of the cDNA phage library, a cDNA synthesiskit, a ZAP-cDNA synthesis kit, and a ZAP-cDNA gigapack III gold cloningkit (STRATAGENE) were used and the library was prepared according toprotocols included with the kit. The size of the thus prepared cDNAphage library was 7.73×10⁵ pfu/ml.

(2) Screening of cDNA Library Using Serum

Immunoscreening was carried out using the above-prepared dog testis cDNAphage library. Specifically, host Escherichia coli (XL1-Blue MRF′) wasinfected with the phage so that 2210 clones were present on a φ90×15 mmNZY agarose plate. Cells were cultured at 42° C. for 3 to 4 hours, so asto cause plaque formation. The plate was covered with a nitrocellulosemembrane (Hybond C Extra: GE HealthCare Bio-Sciences) impregnated withIPTG (isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours. Proteinswere induced, expressed, and then transferred to the membrane.Subsequently, the membrane was recovered, immersed, and shaken in TBS(10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% powdered skim milkat 4° C. overnight, so that nonspecific reaction was suppressed. Thefilter was caused to react with 500-fold diluted sera of dogs withcancer at room temperature for 2 to 3 hours.

As the above sera from dogs with cancer, sera collected from dogs withbreast cancer were used. The sera were stored at −80° C. and thensubjected to pretreatment immediately before use. Pretreatment for serawas performed by the following method. Specifically, host Escherichiacoli (XL1-Blure MRF′) was infected with X, ZAP Express phage into whichno foreign gene had been inserted, and then cultured on NZY plate mediumat 37° C. overnight. Subsequently, a 0.2 M NaHCO₃ buffer (pH 8.3)containing 0.5 M NaCl was added to the plate and then the plate was leftto stand at 4° C. for 15 hours. The supernatants were collected asEscherichia coli/phage extracts. Next, the collected Escherichiacoli/phage extract was passed through a NHS-column (GE HealthCareBio-Sciences), so as to immobilize the Escherichia coli.phage-derivedprotein. The serum of a dog with cancer was passed through the column towhich the protein had been immobilized for reaction, thereby removingEscherichia coli and antibodies adsorbed to the phage from the serum.Each serum fraction that had passed through the column was diluted500-fold with TBS containing 0.5% powdered skim milk, and the resultantwas used as an immunoscreening material.

A membrane, to which the thus treated serum and the fusion protein hadbeen blotted, was washed 4 times with TBS-T (0.05% Tween20/TBS). Themembrane was reacted with goat anti-dog IgG (Goat anti Dog IgG-h+I HRPconjugated: BETHYL Laboratories) diluted 5000-fold as a secondaryantibody with TBS containing 0.5% powdered skim milk at room temperaturefor 1 hour. Detection was carried out by enzyme color reaction using anNBT/BCIP reaction solution (Roche). Colonies corresponding to the colorreaction positive site were collected from the φ90×15 mm NZY agaroseplate, and then dissolved in 500 μl of SM buffer (100 mM NaCl, 10 mMMgClSO₄, 50 mM Tris-HCl, 0.01% gelatin, pH 7.5). Until unification ofcolor reaction positive colonies, secondary screening and tertiaryscreening were repeated by a method similar to the above. Thus, 30940phage clones that had reacted with serum IgG were screened so that 5positive clones were isolated.

(3) Homology Search for Isolated Antigen Gene

A procedure for conversion of phage vectors to plasmid vectors wasperformed for the 5 positive clones isolated by the above method for thepurpose of subjecting the clones to nucleotide sequence analysis.Specifically, 200 μl of a solution of host Escherichia coli (XL1-BlueMRF′) prepared to give an absorbance OD₆₀₀ of 1.0, 250 μl of a purifiedphage solution, and 1 μl of ExAssist helper phage (STRATAGENE) weremixed and allowed to react at 37° C. for 15 minutes. After that, 3 ml ofLB medium was added, cells were cultured at 37° C. for 2.5 to 3 hours,and then the resultant was immediately put in water bath at 70° C. forincubation for 20 minutes. Centrifugation was carried out at 4° C.,1000×g for 15 minutes, and then the supernatant was collected as aphagemid solution. Subsequently, 200 μl of a solution prepared fromphagemid host Escherichia coli SOLR to give an absorbance OD₆₀₀ of 1.0and 10 μl of the purified phage solution were mixed, followed by 15minutes of reaction at 37° C. 50 μl of the resultant was plated on LBagar medium containing ampicillin (at final concentration of 50 μg/ml)and then cultured overnight at 37° C. A single colony of transformedSOLR was collected and then cultured on LB medium containing ampicillin(at final concentration of 50 μg/ml) at 37° C. After culture, plasmidDNA carrying an insert of interest was purified using a QIAGEN plasmidMiniprep Kit (QIAGEN).

The purified plasmid was subjected to the analysis of the entiresequence of the insert by the primer walking method using the T3 primerof SEQ ID NO: 31 and the T7 primer of SEQ ID NO: 32. The gene sequencesof SEQ ID NOS: 5, 7, 9, 11, and 13 were obtained by the sequenceanalysis. With the use of the nucleotide sequences of the genes and theamino acid sequences thereof (SEQ ID NOS: 6, 8, 10, 12, and 14),homology search program BLAST search(http://www.ncbi.nlm.nih.gov/BLAST/) was conducted for searchinghomology with known genes. As a result, it was revealed that all thefive obtained genes were genes encoding CAPRIN-1. The sequenceidentities among the five genes were 100% at the nucleotide sequencelevel and 99% at the amino acid sequence level in the regions to betranslated into proteins. The sequence identities of these genes and thehuman homologue-encoding gene were 94% at the nucleotide sequence leveland 98% at the amino acid sequence level in the regions to be translatedinto proteins. The nucleotide sequences of the human homologues arerepresented by SEQ ID NOS: 1 and 3 and the amino acid sequences of thesame are represented by SEQ ID NOS: 2 and 4. Also, the sequenceidentities of the obtained dog genes and the cattle homologue-encodinggene were 94% at the nucleotide sequence level and 97% at the amino acidsequence level in the regions to be translated into proteins. Thenucleotide sequence of the cattle homologue is represented by SEQ ID NO:15 and the amino acid sequence of the same is represented by SEQ ID NO:16. In addition, the sequence identities of the human homologue-encodinggenes and the cattle homologue-encoding gene were 94% at the nucleotidesequence level and 93% to 97% at the amino acid sequence level in theregions to be translated into proteins. Also, the sequence identities ofthe obtained dog genes and the horse homologue-encoding gene were 93% atthe nucleotide sequence level and 97% at the amino acid sequence levelin the regions to be translated into proteins. The nucleotide sequenceof the horse homologue is represented by SEQ ID NO: 17 and the aminoacid sequence of the same is represented by SEQ ID NO: 18. In addition,the sequence identities of the human homologue-encoding genes and thehorse homologue-encoding gene were 93% at the nucleotide sequence leveland 96% at the amino acid sequence level in the regions to be translatedinto proteins. Also, the sequence identities of the obtained dog genesand the mouse homologue-encoding genes were 87% to 89% at the nucleotidesequence level and 95% to 97% at the amino acid sequence level in theregions to be translated into proteins. The nucleotide sequences of themouse homologues are represented by SEQ ID NOS: 19, 21, 23, 25, and 27and the amino acid sequences of the same are represented by SEQ ID NOS:20, 22, 24, 26, and 28. In addition, the sequence identities of thehuman homologue-encoding genes and the mouse homologue-encoding geneswere 89% to 91% at the nucleotide sequence level and were 95% to 96% atthe amino acid sequence level in the regions to be translated intoproteins. Also, the sequence identities of the obtained dog genes andthe chicken homologue-encoding gene were 82% at the nucleotide sequencelevel and 87% at the amino acid sequence level in the regions to betranslated into proteins. The nucleotide sequence of the chickenhomologue is represented by SEQ ID NO: 29 and the amino acid sequence ofthe same is represented by SEQ ID NO: 30. In addition, the sequenceidentities of the human homologue-encoding genes and the chickenhomologue-encoding gene were 81% to 82% at the nucleotide sequence leveland 86% at the amino acid sequence level in the regions to be translatedinto proteins.

(4) Gene Expression Analysis in Each Tissue

The expression of genes obtained by the above method was examined in dogand human normal tissues and various cell lines by an RT-PCR method.Reverse transcription reaction was performed as follows. Specifically,total RNA was extracted from 50 mg to 100 mg of the tissue or 5 to10×10⁶ cells of the cell line using a TRIZOL reagent (Invitrogen)according to the accompanying protocols. cDNA was synthesized with thetotal RNA using a Superscript First-Strand Synthesis System for RT-PCR(Invitrogen) according to the accompanying protocols. PCR was performedas follows using primers of SEQ ID NOS: 33 and 34 specific to theobtained genes. Specifically, reagents and an accompanying buffer wereadded to 0.25 μl of the sample prepared by the reverse transcriptionreaction to a total volume of 25 μl, so that the resultant contained theabove primers of 2 μM each, dNTPs of 0.2 mM each, and 0.65 U ExTaqpolymerase (Takara Shuzo Co., Ltd.). PCR was carried out by repeating acycle of 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 30seconds 30 times using a Thermal Cycler (BIO RAD). The abovegene-specific primers are capable of amplifying the region ranging fromnucleotides 206 to 632 in the nucleotide sequence of SEQ ID NO: 5 (dogCAPRIN-1 gene) and the region ranging from nucleotides 698 to 1124 inthe nucleotide sequence of SEQ ID NO: 1 (human CAPRIN-1 gene). As acontrol for comparison, GAPDH-specific primers of SEQ ID NOS: 35 and 36were also used concurrently. As a result, as shown in FIG. 1, strongexpression was observed in testis among normal dog tissues, whileexpression was observed in dog breast cancer and adenocarcinoma tissues.Moreover, the observation of the expression of the human homologues fromthe obtained genes was also carried out. As a result, similarly to thecase of the dog CAPRIN-1 gene, expression could be observed in onlytestis among normal tissues. However, in the case of cancer cells,expression was detected in many types of cancer cell lines, includingbreast cancer, brain tumor, leukemia, lung cancer, and esophageal cancercell lines. Expression was observed particularly in many breast cancercell lines. It was confirmed by the results that the expression ofCAPRIN-1 is not observed in normal tissues other than testis, whileCAPRIN-1 was expressed in many cancer cells and particularly in breastcancer cell lines.

In FIG. 1, reference number 1 on each vertical axis indicates theexpression patterns of genes identified above and reference number 2indicates the expression patterns of the GAPDH gene as a control.

(5) Immunohistochemical Staining (5)-1 CAPRIN-1 Expression in Mouse andDog Normal Tissues

Mice (Balb/c, female) and dogs (beagles, female) were exsanguinatedunder ether anesthesia and ketamine/isoflurane anesthesia. Afterlaparotomy, each organ (stomach, liver, eyeball, thymus gland, muscle,bone marrow, uterus, small bowel, esophagus, heart, kidney, salivarygland, large bowel, mammary gland, brain, lung, skin, adrenal gland,ovary, pancreas, spleen, and bladder) was transferred to a 10-cm dishcontaining PBS. Each organ was cut open in PBS and then subjected toperfusion fixation overnight in 0.1 M phosphate buffer (pH 7.4)containing 4% paraformaldehyde (PFA). The perfusion solution wasdiscarded, the tissue surface of each organ was rinsed with PBS, a PBSsolution containing 10% sucrose was added to a 50-ml centrifuge tube,each tissue was added to the tube, and then the tube was shaken using arotor at 4° C. for 2 hours. The solution was replaced by a PBS solutioncontaining 20% sucrose, and then left to stand at 4° C. until the tissuesank. The solution was replaced by a PBS solution containing 30% sucroseand then left to stand at 4° C. until the tissue sank. The tissue wasremoved and then needed portions were excised with a surgical scalpel.Next, an OCT compound (Tissue Tek) was added to the tissue so that itwas thoroughly applied to the tissue surface, and then the tissue wasplaced in a cryomold. The cryomold was placed on dry ice for quickfreezing. Thereafter, the tissue was sliced to 10 μm to 20 μm using acryostat (LEICA). Slices were air-dried on slide glasses using a hairdryer for 30 minutes, to prepare the sliced tissue mounted on a slideglass. Next, each sample was placed in a staining bottle filled withPBS-T (saline containing 0.05% Tween20) and then subjected toreplacement with PBS-T being repeated three times every 5 minutes.Excess water around the sections was removed with Kimwipes, and then thesections were circled using a DAKOPEN (DAKO). As blocking solutions, anMOM mouse Ig blocking reagent (VECTASTAIN) and a PBS-T solutioncontaining 10% FBS were overlaid on mouse tissue and dog tissue,respectively, and then left to stand in a moist chamber at roomtemperature for 1 hour. Next, a solution of the anti-CAPRIN-1 monoclonalantibody (monoclonal antibody #1 prepared in Example 4) of 10 μg/mladjusted with a blocking solution, which reacts with cancer cellsurfaces and comprises the heavy chain variable region of SEQ ID NO: 42and the light chain variable region of SEQ ID NO: 46, was placed on andthen left to stand overnight in a moist chamber at 4° C. 10 minutes ofwashing with PBS-T was performed 3 times, and then an MOM biotin-labeledanti-IgG antibody (VECTASTAIN) diluted 250-fold with the blockingsolution was placed and then left to stand at room temperature for 1hour in a moist chamber. After ten (10) minutes of washing with PBS-Twas performed 3 times, an avidin-biotin ABC reagent (VECTASTAIN) wasplaced on, and then the sample was left to stand in a moist chamber atroom temperature for 5 minutes. After ten (10) minutes of washing withPBS-T was performed 3 times, a DAB coloring solution (DAB 10 mg+30% H₂O₂10 μl/0.05 M Tris-HCl (pH 7.6) 50 ml) was placed on, and then the samplewas left to stand in a moist chamber at room temperature for 30 minutes.After rinsing with distilled water, a hematoxylin reagent (DAKO) wasplaced on, the sample was left to stand at room temperature for 1minute, and then rinsed with distilled water. The slide glass wasimmersed in 70%, 80%, 90%, 95%, and then 100% ethanol solutions in suchorder for 1 minute each and then left to stand overnight in xylene. Theslide glass was removed, sealed in Glycergel Mounting Medium (DAKO), andthen observed. As a result, the expression of CAPRIN-1 was slightlyobserved within cells of each tissue of salivary gland, kidney, colon,and stomach, but the expression of the same was not observed on cellsurfaces. Furthermore, no expression was observed in tissues from otherorgans.

(5)-2 CAPRIN-1 Expression in Dog Breast Cancer Tissue

Frozen section slides were prepared by a method similar to the aboveusing 108 frozen breast cancer tissue specimens of dogs pathologicallydiagnosed as having malignant breast cancer, and immunohistochemicalstaining was performed using the monoclonal antibody #1 prepared inExample 4. As a result, the expression of CAPRIN-1 was observed in 100out of 108 specimens (92.5%) and CAPRIN-1 was strongly expressed on thesurfaces of cancer cells with a particularly high grade of atypism.

(5)-3 CAPRIN-1 Expression in Human Breast Cancer Tissues

Immunohistochemical staining was performed using 188 breast cancertissue specimens on a paraffin-embedded human breast cancer tissue array(BIOMAX). After 3 hours of treatment of the human breast cancer tissuearray at 60° C., the array was placed in a staining bottle filled withxylene, followed by xylene replacement being repeated three times every5 minutes. Next, a similar procedure was performed with ethanol andPBS-T instead of xylene. The human breast cancer tissue array was placedin a staining bottle filled with 10 mM citrate buffer (pH 6.0)containing 0.05% Tween20. After 5 minutes of treatment at 125° C., thearray was left to stand at room temperature for 40 minutes or more.Excess water around the sections was removed with Kimwipes, the sectionswere circled with a DAKOPEN, and Peroxidase Block (DAKO) was addeddropwise in appropriate amounts. After left to stand at room temperaturefor 5 minutes, the array was placed in a staining bottle filled withPBS-T, followed by PBS-T replacement being repeated three times every 5minutes. As a blocking solution, a PBS-T solution containing 10% FBS wasplaced on the array, and then the array was left to stand in a moistchamber at room temperature for 1 hour. Next, a solution of themonoclonal antibody #1 of 10 μg/ml adjusted with a PBS-T solutioncontaining 5% FBS, which reacts with cancer cell surfaces and had beenprepared in Example 4, was placed on, and the array was left to standovernight in a moist chamber at 4° C. After ten (10) minutes of washingwith PBS-T was performed 3 times, Peroxidase Labeled Polymer Conjugated(DAKO) was added dropwise in appropriate amounts and then the array wasleft to stand in a moist chamber at room temperature for 30 minutes.After ten (10) minutes of washing with PBS-T was performed 3 times, aDAB coloring solution (DAKO) was placed on and then it was left to standat room temperature for about 10 minutes. The coloring solution wasdiscarded, 10 minutes of washing with PBS-T was performed 3 times, andthen it was rinsed with distilled water. The array was immersed in 70%,80%, 90%, 95%, and then 100% ethanol solutions in such order for 1minute each, and then left to stand in xylene overnight. The slide glasswas removed, sealed in Glycergel Mounting Medium (DAKO), and thenobserved. As a result, the strong expression of CAPRIN-1 was observed in138 out of a total of 188 breast cancer tissue specimens (73%).

(5)-4 CAPRIN-1 Expression in Human Malignant Brain Tumor

Immunohistochemical staining was performed according to a method similarto that used in (5)-3 above with 247 malignant brain tumor tissuespecimens on a paraffin-embedded human malignant brain tumor tissuearray (BIOMAX), using the monoclonal antibody #1 prepared in Example 4.As a result, the strong expression of CAPRIN-1 was observed in 227 outof a total of 247 malignant brain tumor tissue specimens (92%).

(5)-5 CAPRIN-1 Expression in Human Breast Cancer Metastasized Lymph Node

Immunohistochemical staining was performed according to a method similarto that in (5)-3 above with 150 breast cancer metastasized lymph nodetissue specimens on a paraffin-embedded human breast cancer metastasizedlymph node tissue array (BIOMAX), using the monoclonal antibody #1prepared in Example 4. As a result, the strong expression of CAPRIN-1was observed in 136 out of a total of 150 breast cancer metastasizedlymph node tissue specimens (90%). Specifically, it was revealed thatCAPRIN-1 was strongly expressed also in cancer tissues that hadmetastasized from breast cancer.

(5)-6 CAPRIN-1 Expression in Various Human Cancer Tissues

Immunohistochemical staining was performed according to a method similarto the above with specimens on various paraffin-embedded human cancertissue arrays (BIOMAX), using the monoclonal antibody #1 prepared inExample 4. As a result, the strong expression of CAPRIN-1 was observedin esophageal cancer, colon cancer, rectal cancer, lung cancer, renalcancer, bladder cancer, and uterine cervix cancer.

Example 2 Preparation of Novel Human Cancer Antigen Protein (1)Preparation of Recombinant Protein

Based on the gene of SEQ ID NO: 1 obtained in Example 1, a recombinantprotein from the human homologous gene was prepared by the followingmethod. PCR was performed in a total volume of 50 μl with 1 μl of cDNA,two primers (SEQ ID NOS: 37 and 38 comprising Sac I and Xho Irestriction enzyme cleavage sequences) of 0.4 μM each, 0.2 mM dNTP, and1.25U PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.), prepared byadding the reagents and an accompanying buffer. The expression had beenconfirmed by an RT-PCR method for the cDNA used herein from amongvarious tissue.or cell-derived cDNAs prepared in Example 1. PCR waspreformed by repeating a cycle of 98° C. for 10 seconds and 68° C. for2.5 minutes 30 times using a Thermal Cycler (BIO RAD). The above twoprimers are capable of amplifying a region encoding the entire aminoacid sequence of SEQ ID NO: 2. After PCR, the thus amplified DNA wassubjected to electrophoresis on 1% agarose gel, and then an about 2.1kbp DNA fragment was purified using a QIAquick Gel Extraction Kit(QIAGEN).

The thus purified DNA fragment was ligated to a cloning vector PCR-Blunt(Invitrogen). After transformation of Escherichia coli with it, plasmidwas collected. It was verified by sequencing that the thus amplifiedgene fragment has the sequence of interest. The plasmid having a matchedsequence with the sequence of interest was treated with Sac I and Xho Irestriction enzymes and then purified with a QIAquick Gel ExtractionKit. The gene sequence of interest was inserted into an Escherichia coliexpression vector pET30a (Novagen) treated with Sac I and Xho Irestriction enzymes. A His-tag fused recombinant protein can be producedusing the vector. The plasmid was transformed into Escherichia coli forexpression, BL21(DE3), and then expression was induced with 1 mM IPTG,so that the protein of interest was expressed in Escherichia coli.

(2) Purification of Recombinant Protein

The above-obtained recombinant Escherichia coli expressing the gene ofSEQ ID NO: 1 was cultured in LB medium containing 30 μg/ml kanamycin at37° C. until absorbance at 600 nm reached around 0.7,isopropyl-β-D-1-thiogalactopyranoside was added at a final concentrationof 1 mM, and then cells were cultured at 37° C. for 4 hours.Subsequently, centrifugation was performed at 4800 rpm for 10 minutesand then cells were collected. The resulting cell pellet was suspendedin phosphate buffered saline and centrifuged at 4800 rpm for 10 minutes,and then cells were washed.

The cells were suspended in phosphate buffered saline and then disruptedby ultrasonication on ice. The resulting lysate of the ultrasonicatedEscherichia coli was subjected to centrifugation at 6000 rpm for 20minutes, and then the resulting supernatant was regarded as a solublefraction and the precipitate was regarded as an insoluble fraction.

The soluble fraction was added to a nickel chelate column adjustedaccording to a conventional method (carrier: Chelating Sepharose™ FastFlow (GE HealthCare); column capacity of 5 ml; and equilibration buffer:50 mM hydrochloride buffer (pH 8.0)). Unadsorbed fractions were washedoff with 50 mM hydrochloride buffer (pH 8.0) in an amount 10 times thecolumn capacity and 20 mM phosphate buffer (pH 8.0) containing 20 mMimidazole. Immediately after washing, 6 beds were eluted with 20 mMphosphate buffer (pH 8.0) containing 100 mM imidazole. The elution ofthe protein of interest was confirmed by Coomassie staining on theelution fraction with 20 mM phosphate buffer (pH 8.0) containing 100 mMimidazole, and then the elution fraction was added to a strong anionexchange column (carrier: Q Sepharose™ Fast Flow (GE HealthCare); columncapacity of 5 ml; and 20 mM phosphate buffer (pH 8.0) as anequilibration buffer). An unadsorbed fraction was washed off with 20 mMphosphate buffer (pH 7.0) in an amount 10 times the column capacity and20 mM phosphate buffer (pH 7.0) containing 200 mM sodium chloride.Immediately after washing, 5 beds were eluted with 20 mM phosphatebuffer (pH 7.0) containing 400 mM sodium chloride, and thus the purifiedfraction of the protein having the amino acid sequence represented bySEQ ID NO: 2 was obtained.

200 μl of each purified sample obtained by the above method wasdispensed into 1 ml of reaction buffer (20 mM Tris-Hcl, 50 mM, NaCl, 2mM CaCl₂ pH 7.4), followed by addition of 2 μl of enterokinase(Novagen). After that, the resultant was left to stand overnight at roomtemperature for reaction so that His-tag was cleaved off, and thenpurification was performed using an Enterokinase Cleavage Capture Kit(Novagen) according to the accompanying protocols. Next, 1.2 ml of thepurified sample obtained by the above method was subjected to the bufferreplacement with physiological phosphate buffer (Nissui PharmaceuticalCo., Ltd.) using an ultrafiltration NANOSEP 10K OMEGA (PALL). Further,sterile filtration was performed using HT Tuffryn Acrodisc 0.22 μm(PALL) and then the resultant was used for the following experiment.

Example 3 Preparation of Chicken Monoclonal Antibody Against CAPRIN-1

300 μg of the antigen protein (human CAPRIN-1) 0 shown by SEQ ID NO: 2prepared in Example 2 was mixed with an equivalent amount of Freund'scomplete adjuvant, and then this was used as an antigen solution for onechicken. The antigen solution was intraperitoneally administered to7-week-old chickens, and then the administration was performed 7 timesevery 4 weeks, and thus immunization was completed. Each spleen wasexcised on day 4 after the final immunization, and sandwiched betweentwo sterilized slide glasses and then crushed. The resultant was washedwith PBS(−) (Nissui) and then centrifuged at 1500 rpm for 10 minutes toremove the supernatant. This procedure was repeated 3 times, so thatsplenocytes were obtained. The thus obtained splenocytes and chickenmyeloma cells deficient in light chain were mixed at a ratio of 5:1. Theused chicken myeloma cells had been established from a chicken by atransformation using an avian reticuloendotheliosis virus. A PEGsolution prepared by mixing 200 μl of IMDM medium containing 10% FBSheated at 37° C. and 800 μl of PEG1500 (Boehringer) was added to themixture, left to stand for 5 minutes for cell fusion, and then subjectedto centrifugation at 1700 rpm for 5 minutes. After removal of thesupernatant, cells were suspended in 300 ml of IMDM medium containing10% FBS, supplemented with a HAT solution (Gibco) (2% equivalent) (HATselective medium), and then the cell suspension was plated on thirty96-well plates (Nunc) at 100 μl per well. Cells were cultured for 7 daysat 37° C. under conditions of 5% CO₂, so that hybridoma prepared byfusion of splenocytes and chicken myeloma cells were obtained.

Hybridomas were selected using as a marker the binding affinity of theantibody produced by the prepared hybridomas to the CAPRIN-1 protein.The CAPRIN-1 protein solution (1 μg/ml) prepared in Example 2 was addedto a 96-well plate at 100 μl per well and then left to stand at 4° C.for 18 hours. Each well was washed 3 times with PBS-T, 400 μl of a 0.5%Bovine Serum Albumin (BSA) solution (Sigma) was added per well, and thenthe plate was left to stand at room temperature for 3 hours. Thesolution was removed, and then the wells were washed three times with400 μl of PBS-T per well. The culture supernatant of the above-obtainedhybridomas was added at 100 μl per well, and then left to stand at roomtemperature for 2 hours. After washing each well three times with PBS-T,an HRP-labeled anti-chicken IgY antibody (SIGMA) diluted 5000-fold withPBS was added at 100 μl per well and the resultant was then left tostand at room temperature for 1 hour. After washing the wells threetimes with PBS-T, 100 μl of a TMB substrate solution (Thermo) was addedper well and then left to stand for 15 to 30 minutes for coloringreaction. After color development, 100 μl of 1N sulfuric acid was addedper well to stop the reaction, and then absorbances at 450 nm and 595 nmwere measured using an absorption spectrometer. As a result, severalhybridomas producing antibodies with high absorbance values wereselected.

The thus selected hybridomas were added to a 96-well plate at 0.5 cellsper well and then cultured. After 1 week, hybridomas that had formedsingle colonies in wells were observed. These cells in the wells werefurther cultured, and then hybridomas were selected using as a markerthe binding affinity of antibodies produced by the cloned hybridomas tothe CAPRIN-1 protein. The CAPRIN-1 protein solution (1 μg/ml) preparedin Example 2 was added to a 96-well plate at 100 μl per well, and thenleft to stand at 4° C. for 18 hours. Each well was washed with PBS-Tthree times, 400 μl of a 0.5% BSA solution was added per well, and thenthe resultant was left to stand at room temperature for 3 hours. Thesolution was removed, and then the wells were washed three times with400 μl of PBS-T per well. 100 μl of each culture supernatant of theabove-obtained hybridomas was added per well, and then the plate wasleft to stand at room temperature for 2 hours. After washing each wellthree times with PBS-T, 100 μl of an HRP-labeled anti-chicken IgYantibody (SIGMA) diluted 5000-fold with PBS was added per well and thenleft to stand at room temperature for 1 hour. After washing the wellsthree times with PBS-T, 100 μl of a TMB substrate solution (Thermo) wasadded per well, and then left to stand for 15 to 30 minutes for coloringreaction. After color development, 100 μl of 1N sulfuric acid was addedper well to stop the reaction and then absorbances at 450 nm and 595 nmwere measured using an absorption spectrometer. As a result, severalhybridoma cell lines producing monoclonal antibodies reactive with theCAPRIN-1 protein were obtained.

Next, of those monoclonal antibodies, antibodies reactive with the cellsurfaces of breast cancer cells expressing CAPRIN-1 were selected.Specifically, 5×10⁵ cells of the human breast cancer cell lineMDA-MB-231V were subjected to centrifugation in a 1.5-ml microcentrifugetube, and 100 μl of the culture supernatant of each of the abovehybridomas was added to the tube, and then the tube was left to stand onice for 1 hour. After washing with PBS, an FITC-labeled goatanti-chicken IgG (H+L) antibody (SouthernBiotech) diluted 30-fold withPBS containing 0.1% FBS was added, and then the resultant was left tostand on ice for 1 hour. After washing with PBS, fluorescence intensitywas measured using a FACS caliber (Becton, Dickinson and Company).Meanwhile, procedures similar to the above were performed for medium forculturing hybridomas, so that a control sample was obtained. As aresult, one monoclonal antibody (monoclonal antibody #1) that hadexhibited fluorescence intensity stronger than that of the control, andthat is, that reacted with the cell surfaces of CAPRIN-1-expressingbreast cancer cells, was selected.

Example 4 Characterization of Selected Antibody (1) Cloning of Genes ofAnti-CAPRIN-1 Monoclonal Antibody Variable Regions

mRNA was extracted from a chicken-derived hybridoma cell line producingmonoclonal antibodies (selected in Example 3) reactive with the surfacesof CAPRIN-1-expressing breast cancer cells. An RT-PCR method usingprimers specific to the chicken FR1-derived sequence and the chickenFR4-derived sequence was performed therefor, and the gene of the heavychain variable (VH) region and the gene of the light chain variable (VL)region of the antibody were obtained. mRNA was also extracted from twomouse-derived hybridoma cell lines producing monoclonal antibodiesreactive with the surfaces of CAPRIN-1-expressing breast cancer cells.An RT-PCR method using primers specific to the mouse FR1-derivedsequence and the mouse FR4-derived sequence was performed therefor, andthe gene of the heavy chain variable (VH) region and the gene of thelight chain variable (VL) region of each antibody were obtained. Forsequence determination, these genes were cloned into a pCR2.1 vector(Invitrogen).

(1)-1 RT-PCR

After extraction of total RNA from 10⁶ cells of each hybridoma cell lineusing a High Pure RNA Isolation Kit (Roche), cDNA was synthesized usinga PrimeScriptII 1st strand cDNA Synthesis Kit (Takara). These procedureswere performed according to protocols attached to each kit.

The gene of the chicken antibody heavy chain variable region and thegene of the chicken antibody light chain variable region, and the geneof the mouse antibody heavy chain variable region and the gene of themouse antibody light chain variable region were each amplified by a PCRmethod according to a conventional method using the thus synthesizedcDNA as a template and KOD-Plus-DNA Polymerase (TOYOBO).

To obtain the gene of the chicken antibody VH region, a primer specificto the chicken heavy chain FR1 sequence and a primer specific to thechicken heavy chain FR4 sequence were used. Furthermore, to obtain thegene of the VL region, a primer specific to the chicken light chain FR1sequence and a primer specific to the chicken light chain FR4 were used.The genes of mouse antibody VH and VL regions were obtained in a mannersimilar to the above. Specifically, a primer specific to the mouse heavychain FR1 sequence, a primer specific to the mouse heavy chain FR4sequence, a primer specific to the mouse light chain FR1 sequence, and aprimer specific to the mouse light chain FR4 were used.

The thus obtained PCR products were each subjected to agarose gelelectrophoresis, and DNA bands of the VH region and the VL region wereexcised. DNA fragments were purified using a QIAquick Gel purificationkit (QIAGEN) according to the accompanying protocols. The purified DNAwas cloned into a pCR2.1 vector using a TA cloning kit (Invitrogen). Theligated vector was transformed into DH5 competent cells (TOYOBO)according to a conventional method. 10 clones of each transformant werecultured overnight in medium (100 μg/ml ampicillin) at 37° C., and thenplasmid DNA was purified using a Qiaspin Miniprep kit (QIAGEN).

(1)-2 Sequence Determination

The gene sequences of the VH region and the VL region in each plasmidobtained above were analyzed with an M13 forward primer (SEQ ID NO: 47)and an M13 reverse primer (SEQ ID NO: 48) on a fluorescence sequencer(DNA sequencer 3130XL; ABI), using a Big Dye Terminator Ver3.1 CycleSequencing Kit (ABI) according to the accompanying protocols. As aresult, each gene sequence was determined. The sequences were identicalamong the 10 clones.

The thus obtained gene sequence encoding the heavy chain variable regionof the chicken-derived monoclonal antibody is represented by SEQ ID NO:49 and the amino acid sequence thereof is represented by SEQ ID NO: 42,and the thus obtained gene sequence encoding the light chain variableregion is represented by SEQ ID NO: 50 and the amino acid sequencethereof is represented by SEQ ID NO: 46.

Specifically, it was revealed that the monoclonal antibody #1 comprisesthe heavy chain variable region of SEQ ID NO: 42 and the light chainvariable region of SEQ ID NO: 46, wherein CDR1, CDR2, and CDR3 in theheavy chain variable region consist of the amino acid sequences of SEQID NOS: 39, 40, and 41, respectively, and the CDR1, CDR2, and CDR3 inthe light chain variable region consist of the amino acid sequences ofSEQ ID NOS: 43, 44, and 45, respectively.

(2) Preparation of Human-Chicken Chimeric Recombinant Antibody andMouse-Chicken Chimeric Antibody

An amplification fragment of the gene of the heavy chain variable region(SEQ ID NO: 42) of the chicken monoclonal antibody #1 obtained in (1)above was treated at both ends with restriction enzymes and thenpurified. The resulting fragment was inserted into a pcDNA4/myc-His(Invitrogen) vector according to a conventional method, into which achicken antibody-derived leader sequence comprising SEQ ID NO: 51 and ahuman IgG1 H-chain constant region comprising SEQ ID NO: 52 had alreadybeen inserted. Furthermore, an amplification fragment of the gene of thechicken monoclonal antibody #1 light chain variable region (SEQ ID NO:46) was treated at both ends with restriction enzymes and then purified.The resulting fragment was inserted into a pcDNA3.1/myc-His (Invitrogen)vector according to a conventional method, into which a chickenantibody-derived leader sequence comprising SEQ ID NO: 51 and a humanIgG1 L-chain constant region comprising SEQ ID NO: 53 had already beeninserted.

Next, the above recombinant vector into which the heavy chain variableregion (SEQ ID NO: 42) of the chicken monoclonal antibody #1 had beeninserted, and the above recombinant vector into which the chickenmonoclonal antibody #1 light chain variable region (SEQ ID NO: 46) hadbeen inserted, were introduced into CHO-K1 cells (obtained from RIKENCell Bank). Specifically, 2×10⁵ CHO-K1 cells cultured in 1 ml of Ham'sF12 medium (Invitrogen) containing 10% FBS per well of a 12-well cultureplate were washed with PBS(−). 1 ml of Ham's F12 medium containing 10%FBS was further added per well and then a mixture of 250 ng of each ofthe above vectors dissolved in 30 μl of OptiMEM (Invitrogen) and 30 μlof a Polyfect transfection reagent (QIAGEN) was added to each well.CHO-K1 cells into which the above recombinant vector had been introducedwere cultured in Ham's F12 medium containing 10% FBS, supplemented with200 μg/ml Zeocin (Invitrogen) and 200 μg/ml geneticin (Roche). CHO-K1cells into which the above recombinant vector had been introduced wereplated in a 96-well plate at 0.5 cells per well. Thus, a cell linestably producing a human-chicken chimeric antibody #1 (also referred toas #1) having the chicken monoclonal antibody #1 variable region wasprepared. The thus prepared cell line was cultured in a 150 cm² flaskcontaining 30 ml of serum-free OptiCHO medium (Invitrogen) at 5×10⁵cells/ml for 5 days. Then, a culture supernatant containing #1 wasobtained.

With a method similar to the above, an amplification fragment of thegene of the heavy chain variable region (SEQ ID NO: 42) of the chickenmonoclonal antibody #1 was treated at both ends with restriction enzymesand then purified. The resultant was inserted according to aconventional method into a pcDNA4/myc-His (Invitrogen) vector into whicha chicken antibody-derived leader sequence and a mouse IgG1 H-chainconstant region had already been inserted. Furthermore, an amplificationfragment of the gene of the light chain variable region (SEQ ID NO: 46)of the chicken monoclonal antibody #1 was treated at both ends withrestriction enzymes and then purified. The resultant was then insertedaccording to a conventional method into a pcDNA3.1/myc-His (Invitrogen)vector into which a chicken antibody-derived leader sequence and themouse IgG1 L-chain constant region had already been inserted. Theresultant was introduced into CHO-K1 cells in a manner similar to theabove and thus a cell line stably producing a mouse-chicken chimericantibody #1 comprising the chicken monoclonal antibody #1 variableregion was prepared. The cells were cultured at 5×10⁵ cells/ml using a150 cm² flask and 30 ml of serum free OptiCHO medium (Invitrogen) for 5days, so that a culture supernatant containing the mouse-chickenchimeric antibody #1 was obtained.

(3) CAPRIN-1 Expression on Various Cancer Cell Surfaces UsingAnti-CAPRIN-1 Antibody #1

Next, 7 breast cancer cell lines (MDA-MB-157, T47D, MRK-nu-1,MDA-MB-231V, BT20, SK-BR-3, and DA-MB-231T) for which CAPRIN-1 geneexpression had been observed, and the other 3 breast cancer cell lines(MDA-MB-231C, MCF-7, and ZR75-1), 5 glioma cell lines (T98Q SNB19, U251,U87MG, and U373), 4 renal cancer cell lines (Ca Caki-2, A498, and ACHN),2 gastric cancer cell lines (MNK28 and MNK45), 5 colorectal cancer celllines (HT29, LoVo, Caco2, SW480, and HCT116), 3 lung cancer cell lines(A549, QG56, and PC8), 4 leukemia cell lines (AML5, Namalwa, BDCM,RPI1788), one lymphoma cell line (Ramos), one uterine cervix cancer cellline (SW756), one bladder cancer cell line (T24), and one esophagealcancer cell line (KYSE180) were examined for CAPRIN-1 protein expressionon the cell surfaces of each cell line using the culture supernatants ofCHO-K1 cells containing #1 obtained in (2) above. 10⁶ cells of each cellline were centrifuged in a 1.5 ml microcentrifuge tube. Each cellculture supernatant (100 μl) of CHO-K1 cells containing #1 obtained in(2) above was added and then left to stand on ice for 1 hour. Afterwashing with PBS, a FITC-labeled goat-anti human IgG (H+L) antibody(SouthernBiotech) and a FITC-labeled anti-mouse IgG (H+L) antibody(Invitrogen) diluted 500-fold with PBS containing 0.1% FBS were addedand then left to stand on ice for 1 hour. After washing with PBS,fluorescence intensity was measured using a FACS Calibur (Becton,Dickinson and Company). Meanwhile, procedures similar to the above wereperformed using a culture supernatant of CHO-K1 cells into which noantibody gene had been introduced and medium for culturing hybridomas,so that a negative control sample was prepared. As a result, cells towhich the antibody #1 had been added exhibited fluorescence intensitystronger by 20% or more than that of the control. Specifically,fluorescence intensity was enhanced to 4900% in the case of SK-BR-3 and5000% in the case of MDA-MB-231V. It was revealed by these results thatthe CAPRIN-1 protein was expressed on the cell membrane surfaces of theabove human cancer cell lines. The percentage of enhancement in theabove fluorescence intensity was expressed as percentage of increase inmean fluorescence intensity (MFI level) in each type of cell andcalculated by the following formula.

Percentage of increase in mean fluorescence intensity(percentage ofenhancement in fluorescence intensity)(%)=((MFI level in cells havingreacted with anti-human CAPRIN-1 antibody)−(MFI level of thecontrol))/(MFI level of control)×100.

(4) Anti-Tumor Effects (ADCC Activity) of Anti-CAPRIN-1 Antibody #1Against Cancer Cells

Next, the anti-CAPRIN-1 antibody #1 was evaluated for the cytotoxicactivity (ADCC activity) against cancer cells. Each cell culturesupernatant producing #1 obtained in (2) above was purified using HitrapProteinA Sepharose FF (GE HealthCare), subjected to buffer replacementwith PBS(−), and then filtered with a 0.22 μm filter (Millipore). Theresultants were used as antibodies for activity measurement. 10⁶ cellsof MDA-MB-157 human breast cancer cell line were collected in a 50-mlcentrifuge tube, 100 μCi chromium-51 was added, and then incubation wasperformed at 37° C. for 2 hours. Subsequently, the resultant was washedthree times with RPMI1640 medium containing 10% FBS. Cells were added toa 96-well V-bottom plate at 5×10³ cells per well for use as targetcells. The above purified antibodies were added to the cells at a finalconcentration of 1 μg/ml. 2.5×10⁵ cells of lymphocytes separated fromhuman peripheral blood according to a conventional method were furtheradded and then cultured for 4 hours at 37° C. under conditions of 5%CO₂. After culture, the amount of chromium-51 released from damagedcancer cells in a culture supernatant was measured, and the ADCCactivity of each anti-CAPRIN-1 antibody against cancer cells wascalculated. As negative control samples, a sample prepared by adding PBSinstead of the anti-CAPRIN-1 antibodies and a sample prepared by addingan isotype control antibody instead of anti-CAPRIN-1 antibodies wereused. As a result, the antibody #1 exhibited 30% or more cytotoxicactivity against MDA-MB-157 (see FIG. 2). In contrast, the activity inthe sample prepared by adding PBS as a negative control and the activityin the sample prepared by adding the isotype control antibody as anegative control were 1.1% and 2.0%, respectively. Similarly, theantibody #1 was examined for the ADCC activity against other cancercells including glioma cell lines T98G and U373, lung cancer cell linesA549 and QG56, renal cancer cell lines Caki-1 and ACHN, a uterine cervixcancer cell line SW756, a bladder cancer cell line T24, an esophagealcancer cell line KYSE180, gastric cancer cell lines MNK28 and MNK45, acolorectal cancer cell line SW480, a leukemia cell line AML5, and alymphoma cell line Ramos. As a result, the anti-CAPRIN-1 antibody #1exhibited 16.4% activity against T98G (1.2% in the case of the isotypecontrol), 23.1% against U373 (3.2% in the case of the isotype control),36.1% against A549 (3.0% in the case of the isotype control), 33.5%against QG56 (0.5% in the case of the isotype control), 28.3% againstCaki-1 (2.6% in the case of the isotype control), 25.1% against ACHN(1.6% in the case of the isotype control), 27.9% against SW756 (2.2% inthe case of the isotype control), 25.8% against T24 (2.0% in the case ofthe negative control), 26.7% against KYSE180 (3.0% in the case of theisotype control), 21.5% against MNK28 (1.9% in the case of the isotypecontrol), 23.0% against MNK45 (3.0% in the case of the isotype control),24.0% against SW480 (1.8% in the case of the isotype control), 8.3%against AML5 (1.8% in the case of the isotype control), and 8.0% againstRamos (2.2% in the case of the isotype control). It was demonstrated bythe above results that the thus obtained anti-CAPRIN-1 antibody #1damages various human cancer cells expressing CAPRIN-1.

(5) Anti-Tumor Effects (CDC Activity) of Anti-CAPRIN-1 Antibody #1Against Cancer Cell

Next, anti-CAPRIN-1 antibody #1 was evaluated for its cytotoxic activity(CDC activity) against cancer cells. Blood collected from a rabbit wasadded to an Eppendorf tube, left to stand at room temperature for 60minutes, and then subjected to 5 minutes of centrifugation at 3000 rpm.Thus, serum for measurement of CDC activity was prepared. 10⁶ cells ofhuman breast cancer cells MDA-MB-231V were collected in a 50-mlcentrifuge tube, 100 μCi chromium-51 was added, and then incubation wasperformed at 37° C. for 2 hours. The resultant was washed three timeswith RPMI medium containing 10% FBS. Subsequently, the cells weresuspended in RPMI medium containing the above-prepared rabbit serum(50%), and then added to a 96-well V-bottom plate at 5×10³ cells perwell. The antibody #1 obtained in (5) above was added to the cells at afinal concentration of 1 μg/ml and then cells were cultured for 4 hoursat 37° C. under conditions of 5% CO₂. After culture, the amount ofchromium-51 released from damaged tumor cells in a culture supernatantwas measured, and then the CDC activity against MDA-MB-231V wascalculated. As a result, the antibody #1 exhibited 25% or more CDCactivity. Therefore, it was revealed that #1 can damage cancer cellsexpressing CAPRIN-1 also by CDC activity.

Example 5 In Vivo Anti-Tumor Effects of Anti-CAPRIN-1 Antibody #1 inMice

Next, the thus obtained anti-CAPRIN-1 antibody #1 was evaluated for itsin vivo anti-tumor effects in tumor-bearing mice. Antibodies used hereinwere prepared by column purification of the culture supernatant of eachcell producing #1 in the same manner as described above.

The anti-tumor effects of the antibody #1 was examined usingtumor-bearing mice into which a mouse-derived cancer cell lineexpressing CAPRIN-1 had been transplanted. 4T1 cells (purchased fromATCC) were transplanted subcutaneously to the dorsal region of 20 Balb/cmice (Japan SLC Inc.) at 5×10⁵ cells/mouse. Cancers were allowed to growto reach a size of about 5 mm in diameter. The antibody #1 wasadministered intraperitoneally to 10 tumor-bearing mice from among the20 mice in an amount of 200 μg (in 200 μl) for one mouse. Subsequently,the same amount of the antibody was administered intraperitoneally toeach tumor-bearing mouse 3 times in total within 2 days. Cancer sizeswere measured every day and anti-tumor effects were examined byobservation. Meanwhile, as a control group, PBS (−) was administeredinstead of the antibodies to the remaining 10 tumor-bearing mice. As aresult of the observation of the anti-tumor effects, in the test groupto which the anti-CAPRIN-1 antibody #1 had been administered, cancerswere found to have regressed to about 55% on day 4, about 32% on day 6,about 7% on day 8, and cancers were found to have almost completelyregressed before days 10 to 14, when the tumor volume at the initiationof the antibody administration was designated 100% (see FIG. 3). In thecontrol group to which PBS(−) had been administered, the tumor size wasincreased to about 170%, 270%, 440%, and 670% on days 4, 6, 8, and 11,respectively (see FIG. 3). It was demonstrated by the results that theobtained antibody #1 exhibits strong anti-tumor effects in vivo againstcancer cells expressing CAPRIN-1. The tumor (cancer) size was calculatedas a volume using the formula: length of major axis×length of minoraxis×length of minor axis×0.5.

INDUSTRIAL APPLICABILITY

The antibodies of the present invention are useful for treating and/orpreventing a cancer.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

SEQUENCE LISTING FREE TEXT

SEQ ID NOS: 31-38, 47, and 48: primers

1. A pharmaceutical composition for treating and/or preventing a cancer,comprising an antibody or a fragment thereof as an active ingredientthat has immunological reactivity with a CAPRIN-1 protein, wherein theantibody comprises a heavy chain variable region comprising SEQ ID NOS:39, 40, and 41 and a light chain variable region comprising SEQ ID NOS:43, 44, and 45 or a fragment thereof.
 2. The pharmaceutical compositionaccording to claim 1, wherein the cancer is breast cancer, brain tumor,leukemia, lymphoma, lung cancer, mastocytoma, renal cancer, uterinecervix cancer, esophageal cancer, gastric cancer, bladder cancer, orcolorectal cancer.
 3. The pharmaceutical composition according to claim1 or 2, wherein the antibody is a human antibody, humanized antibody,chimeric antibody, single chain antibody, or bispecific antibody.
 4. Anantibody, which comprises a heavy chain variable region comprising SEQID NOS: 39, 40, and 41 and a light chain variable region comprising SEQID NOS: 43, 44, and 45, and has immunological reactivity with a CAPRIN-1protein.
 5. The antibody according to claim 4, which is a humanantibody, humanized antibody, chimeric antibody, single chain antibody,or bispecific antibody.
 6. A pharmaceutical combination for treatingand/or preventing a cancer, comprising the pharmaceutical composition ofclaim 1 and a pharmaceutical composition containing an antitumor agent.7. A method for treating and/or preventing a cancer, comprisingadministering to a subject the pharmaceutical composition of claim
 1. 8.A method for treating and/or preventing a cancer, comprising usingpharmaceutical compositions of the pharmaceutical combination of claim 6in combination in a subject.